The data reported in this study are available in the CNGB Nucleotide Sequence Archive (CNSA: <https://db.cngb.org/cnsa>; accession number CNP0000696). Control datasets of cell lines deep sequencing and simulated data were available with accession number CNP0000857. Control datasets of negative individuals were available with accession number CNP0000868.

Introduction {#sec004}
============

Echinococcosis is a life-threatening zoonosis caused by *Echinococcus* spp. tapeworms with a complex life cycle involving intermediate and definitive hosts. Definitive hosts of *Echinococcus* spp. tapeworms are mainly carnivores such as dogs, foxes, and wolves, and intermediate hosts are usually ungulates or rodents such as sheep, cattle, and pika \[[@pntd.0008148.ref001]\]. Humans can be accidentally infected and develop echinococcosis \[[@pntd.0008148.ref002]\]. As one of the most neglected diseases, at any given time, echinococcosis is affecting more than one million people globally \[[@pntd.0008148.ref003]--[@pntd.0008148.ref005]\]. Among the species in genus *Echinococcus*, there are two most important ones in terms of public health, *E*. *granulosus* and *E*. *multilocularis*, responsible for cystic echinococcosis (CE) and alveolar echinococcosis (AE) respectively \[[@pntd.0008148.ref006],[@pntd.0008148.ref007]\]. CE is cosmopolitan, with high endemic areas include western China, Central Asia, eastern Africa, South America, and Mediterranean countries, and AE is mainly in the northern hemisphere \[[@pntd.0008148.ref001],[@pntd.0008148.ref007]\].

The diagnosis of echinococcosis is based on clinical findings, imaging and serological test \[[@pntd.0008148.ref007],[@pntd.0008148.ref008]\]. Imaging includes ultrasound, magnetic resonance imaging, and computed tomography, among which ultrasound is most widely used as the basis for screening and clinical diagnosis \[[@pntd.0008148.ref008]\]. Based on ultrasound observations, the World Health Organization Informal Working Group on Echinococcosis classified CE cysts into six types (cystic lesion or CL, and CE1--5) and AE lesions into different PNM types (Parasite lesion, Neighbor organs, Metastases) \[[@pntd.0008148.ref007],[@pntd.0008148.ref008]\]. Imaging techniques provide the clinician with important clinical information including the location, number, size, and stage of the cysts, which are crucial for the diagnosis of echinococcosis \[[@pntd.0008148.ref007],[@pntd.0008148.ref009],[@pntd.0008148.ref010]\]. However, there are some unsolved issues with imaging techniques, especially the most commonly used ultrasound in diagnosing echinococcosis. The foremost problem is the late diagnosis. As the early phase of infection is generally asymptomatic, patients may remain asymptomatic for years, even permanently. Given the long incubation period (5--20 years), echinococcosis is not easy to be diagnosed in the early stage, and many asymptomatic patients are diagnosed by chance \[[@pntd.0008148.ref011]--[@pntd.0008148.ref013]\]. Besides, detecting small cystic lesions is also a challenge in imaging diagnosis of echinococcosis. It is not easy to distinguish echinococcosis cysts from cysts caused by other reasons, such as liver abscesses, Caroli disease, bilomas and cystadenomas \[[@pntd.0008148.ref014]--[@pntd.0008148.ref017]\]. The long incubation period and complex clinical manifestation of the disease also makes clinical findings difficult, and patients with symptoms are advised to undergo imaging and serological test immediately, thus clinical finding is of limited added value for diagnosis \[[@pntd.0008148.ref007]\]. A serological test could serve as an auxiliary diagnostic tool, but its limitations include cross-reactivity and incompetence to differentiate present and past infections \[[@pntd.0008148.ref018]--[@pntd.0008148.ref020]\]. In consideration of the limitations of the existing diagnosis tools, detecting the cell-free DNA (cfDNA) released by *Echinococcus* spp. tapeworms may serve as a biomarker of the etiological agents \[[@pntd.0008148.ref021],[@pntd.0008148.ref022]\].

CfDNA consists of nucleic acid fragments found extracellularly and mainly exists in the bloodstream, urine and other body fluids \[[@pntd.0008148.ref022]\]. It has been widely used in clinical practice such as non-invasive prenatal testing (NIPT) \[[@pntd.0008148.ref023]\], tumor monitoring \[[@pntd.0008148.ref024]\] and pathogen detection \[[@pntd.0008148.ref025]\]. As for parasite cfDNA, the metabolic activities of the parasites and attacks from the host's immune system may cause the parasites' DNA to be released into the host\'s circulatory system, and the possible mechanisms can be summarized as active secretion and passive release \[[@pntd.0008148.ref022]\]. Several parasitic diseases have been successfully detected with cfDNA, including *Plasmodium* \[[@pntd.0008148.ref026]\], *Trypanosoma* \[[@pntd.0008148.ref027]\], *Leishmania* \[[@pntd.0008148.ref028]\], *Schistosoma* \[[@pntd.0008148.ref029]\] and *Wuchereria* spp. \[[@pntd.0008148.ref030]\]. Cell-free *Echinococcus* spp. DNA had already been suggested as a biomarker for echinococcosis \[[@pntd.0008148.ref021]\], and its existence in plasma or serum was proven with PCR-based methods \[[@pntd.0008148.ref031]--[@pntd.0008148.ref033]\], though with rather low sensitivities (20--25%) \[[@pntd.0008148.ref031]--[@pntd.0008148.ref033]\] Low sensitivity prevents further application of using plasma cfDNA in the diagnosis of echinococcosis. The unsatisfactory performance of the previous attempts could be due to three possible reasons. First, it was hypothesized that the cfDNA of the parasite did not enter the blood circulation unless the hydatid cyst(s) ruptured--thus non-existence of the parasite cfDNA in the host blood circulation made this detection method impossible \[[@pntd.0008148.ref031]\]. Secondly, there is cfDNA from the parasite in the blood circulation, but its concentration is too low to be detected by the designed methods. Thirdly, the understanding of the characteristics of the cfDNA in circulation is limiting the application of cfDNA in detecting the parasitic infection. The better knowledge of cfDNA's characteristics in NIPT has facilitated its improvement from molecular-counting based first-generation testing strategy to global adopted size-based diagnostics \[[@pntd.0008148.ref034]\]. There are studies and reviews on the characteristics of cfDNA in different conditions including cancer, pregnancy, and transplantation \[[@pntd.0008148.ref035]\]. A detailed study on the existence, quantity, and characteristics of cell-free *Echinococcus* spp. DNA in echinococcosis patients' plasma is still missing.

The rapid development of high-throughput sequencing techniques made it feasible to sequence cfDNA in research and medical settings. Compared with target-based PCR methods, sequencing can provide more comprehensive information about cfDNA \[[@pntd.0008148.ref025]\]. High-throughput sequencing of cfDNA has been widely used in tumor and prenatal diagnosis, which provides much more detailed information of cfDNA for clinical practice and research \[[@pntd.0008148.ref023],[@pntd.0008148.ref024]\]. We initiated this study to explore the existence, quantity, and characteristics of cell-free *Echinococcus* spp. DNA in the plasma of echinococcosis patients with high-throughput sequencing. We collected plasma samples from clinically diagnosed echinococcosis patients, produced cfDNA sequencing data with high-throughput sequencing technology, and analyzed the massive data with bioinformatics workflow. The results revealed that high-throughput sequencing of plasma cfDNA could serve as a feasible tool for cell-free *Echinococcus* spp. DNA study and improve our understanding of *Echinococcus* spp. infection in the human body.

Materials and methods {#sec005}
=====================

Ethics statement {#sec006}
----------------

This research was reviewed and approved by the Ethics Committee of Second People\'s Hospital of Tibet Autonomous Region (SPHTAR-ERC-1), Center for Disease Control and Prevention of Tibet Autonomous Region Institutional Review Board (TCDCP-IRB001) as well as the Institutional Review Board of Beijing Genomics Institute in Shenzhen (BGI-IRB18157-T1). All samples were collected with written informed consent from adult participants, and minors' informed consent was given by their guardians.

Samples and processing {#sec007}
----------------------

Blood samples from ultrasound-confirmed echinococcosis patients (N = 23) were collected at diagnosis and before any medical treatment. The patients' gender, age, and clinical classification are shown in [Table 1](#pntd.0008148.t001){ref-type="table"}. Type of echinococcosis was classified based on ultrasound observations and classification system of the World Health Organization Informal Working Group on Echinococcosis. Among these patients, 14 subsequently underwent surgical operations to remove the cystic lesions, and 9 received chemotherapy. The only AE case (S1) at the beginning was diagnosed as a cystic lesion with ultrasound examination, and the lesion sample of this case was confirmed with PCR to be *E*. *multilocularis* infection. All blood samples were collected with Ethylenediaminetetraacetic acid (EDTA) tubes. After collection, plasma samples were stored at 4°C and centrifuged at 4°C within four hours. The blood samples were centrifuged at 1600g for 10 min at 4°C, and the plasma was recentrifuged at 16,000g for 10 min at 4°C. After centrifugation, plasma samples were immediately stored at −80°C for further experiments. Samples of the lesion from the 14 surgically treated patients were also collected and stored at −80°C.

10.1371/journal.pntd.0008148.t001

###### Clinical data and sequencing results of each patient.

A total of 23 echinococcosis patients were involved in the study. Plasma samples were performed with ELISA tests and cell-free DNA sequencing. Lesion samples from surgery patients were performed with PCR tests.

![](pntd.0008148.t001){#pntd.0008148.t001g}

  Patient characteristics   Diagnosis characteristics   Cell-free DNA sequencing
  ------------------------- --------------------------- -------------------------- ---------- ----------------------- ---------- ------------- ------------- ------- -------- -----------------------
  S1                        Male                        34                         CL         *E*. *multilocularis*   positive   742,927,842   615,603,566   11140   18.096   *E*. *multilocularis*
  S2                        Female                      61                         CE3        *E*. *granulosus*       positive   281,355,103   246,691,600   2       0.008    *E*. *granulosus*
  S3                        Female                      30                         CE1, CE4   *E*. *granulosus*       positive   250,618,825   227,517,883   3       0.013    *E*. *granulosus*
  S4                        Male                        30                         CE3        *E*. *granulosus*       positive   280,431,941   249,457,697   17      0.068    *E*. *granulosus*
  S5                        Male                        40                         CE3        *E*. *granulosus*       positive   279,312,347   248,762,189   1       0.004    *E*. *granulosus*
  S6                        Female                      29                         CE1        *E*. *granulosus*       positive   308,530,827   274,958,461   4       0.015    *E*. *granulosus*
  S7                        Female                      44                         CE2        *E*. *granulosus*       positive   263,251,023   236,679,083   2       0.008    *E*. *granulosus*
  S8                        Male                        29                         CE2        *E*. *granulosus*       positive   360,137,779   313,971,836   17      0.054    *E*. *granulosus*
  S9                        Male                        15                         CL         *E*. *granulosus*       negative   351,412,640   320,738,789   37      0.115    *E*. *granulosus*
  S10                       Female                      30                         CE2        *E*. *granulosus*       positive   306,363,351   269,830,299   13      0.048    *E*. *granulosus*
  S11                       Female                      43                         CL         *E*. *granulosus*       positive   262,203,537   231,673,428   173     0.747    *E*. *granulosus*
  S12                       Female                      10                         CL         *E*. *granulosus*       positive   231,127,477   205,492,100   1       0.005    *E*. *granulosus*
  S13                       Female                      58                         CE3        *E*. *granulosus*       positive   245,838,225   219,082,144   15      0.068    *E*. *granulosus*
  S14                       Female                      36                         CE1        *E*. *granulosus*       negative   256,759,640   224,126,469   13      0.058    *E*. *granulosus*
  N1                        Female                      46                         CE1, CE4   NA                      positive   244,658,087   205,140,224   116     0.565    *E*. *granulosus*
  N2                        Female                      59                         CE2        NA                      positive   364,203,245   281,310,703   129     0.459    *E*. *granulosus*
  N3                        Male                        35                         CE2, CE4   NA                      positive   289,831,896   171,116,167   367     2.145    *E*. *granulosus*
  N4                        Male                        58                         CE5        NA                      negative   367,706,652   213,450,366   540     2.530    *E*. *granulosus*
  N5                        Male                        14                         CE5        NA                      positive   248,171,648   203,853,580   125     0.613    *E*. *granulosus*
  N6                        Male                        47                         CE5        NA                      negative   211,410,500   153,887,667   234     1.521    *E*. *granulosus*
  N7                        Male                        27                         CE1        NA                      negative   132,535,233   114,252,814   15      0.131    *E*. *granulosus*
  N8                        Female                      49                         CE2        NA                      positive   117,991,568   104,563,714   10      0.096    *E*. *granulosus*
  N9                        Female                      41                         CE4        NA                      negative   83,740,668    73,423,055    18      0.245    *E*. *granulosus*

^a^ Clinical Type: CL, Cystic lesion. CE1-5, Cystic echinococcosis clinical stage.

^b^ PE: Paired-end Reads.

^c^ RPM: Read-Pairs Per Million.

DNA extraction and high-throughput sequencing {#sec008}
---------------------------------------------

Plasma samples stored at -80°C were thawed, and cfDNA was immediately extracted from plasma using the cfDNA isolation kit. To yield high-quality cfDNA, two kits were used for cfDNA extraction according to the volume of plasma. Among the 23 plasma samples ([S5 Table](#pntd.0008148.s005){ref-type="supplementary-material"}), 22 samples with volume 0.2 to 0.6 ml were extracted with MagPure Circulating DNA Mini KF Kit (Magen, Guangzhou, China), and one sample (N4) with volume 2.2 ml was extracted with QIAamp Circulating Nucleic Acid Kit (Qiagen, Hilden, Germany). The quantity and quality of cfDNA were assessed with Bioanalyzer 2100 (Agilent Technologies, Santa Clara, USA). The concentration of cfDNA was quantified by Qubit Fluorometer (Invitrogen, Carlsbad, USA) and Qubit dsDNA HS Assay kit (Invitrogen, Carlsbad, USA) following the manufacturer's instructions. As the average fragment length of cfDNA was very short, the usual fragmentation step for library preparation was skipped. The qualified cfDNA was further used to construct sequencing libraries. The final quantified libraries were sequenced on the BGISEQ-500 platform (MGI, Shenzhen, China).

PCR test of lesion samples {#sec009}
--------------------------

Lesion samples stored at -80°C were thawed, and DNA was extracted with phenol/chloroform methods. The presence of *Echinococcus* spp. tapeworms DNA in the lesion samples was confirmed with PCR assays which were based on the amplification of a fragment within the NADH dehydrogenase subunit 1 (ND1) mitochondrial gene \[[@pntd.0008148.ref036]\]. The specific primers and probes with fluorescence can also be used for qualitatively distinguishing *E*. *granulosus*, *E*. o*multilocularis*, and *E*. *shiquicus* \[[@pntd.0008148.ref036]\]. PCR was assayed in a final volume of 30 ul, with 25 ul of master mix and 5 ul of DNA extract, in the ABI 7500 (Applied Biosystems, America) Real-Time PCR System. The thermal cycling condition was: 2 min at 50°C, 5 min at 95°C, followed by 40 cycles of 15 sec at 95°C and 45 sec at 60°C.

ELISA test of plasma {#sec010}
--------------------

Plasma samples of the patients before any medical treatment were assayed with Echinococcosis ELISA IgG kit (Beijing BGI-GBI Biotech, Beijing, China) according to the manual. Briefly, phosphate buffered saline (PBS) diluted plasma samples (1:10) were added to the plates. The plates were incubated for 30 min at 37°C and then washed five times with the PBS-Tween buffer. Peroxidase-conjugated goat anti-human IgG, diluted 1:2000 in a PBS buffer supplemented with 0.5% Tween-20 and 1.5% BSA, was added to each well and incubated at 37°C for 30 min. Before the addition of the tetramethyl-benzidine (TMB) substrate, the plates were washed five times with the PBS-Tween buffer. The reaction was stopped by adding 2 mol/L H2SO4. The OD450/630 value was measured by a microtiter plate reader. A positive control sample, a negative control sample, and a blank control sample were included on each plate, with the cut-off value for IgG as 0.18.

Database construction {#sec011}
---------------------

Sequences of *Echinococcus* spp. tapeworms were downloaded from the NCBI GenBank database. To reduce sequence contamination and get a high-quality sequence database, all sequences were quality controlled with the following steps. *Echinococcus* spp. sequences from GenBank were chopped into 100 bp short pseudo-reads (step size 50 bp), then mapped to the *Echinococcus* tapeworm common host genome sequences (sheep, cattle, pigs, humans, and mice) with BLASTn \[[@pntd.0008148.ref037]\]. Pseudo-reads with high similarity (identity ≥ 97%, coverage ≥ 92%, and e-value ≤ 1e-5) to the host genome sequences were considered to be from host sequence contamination. These host-contaminated pseudo-reads were located to their original chopped sequence regions, and then the regions were masked with BEDTools \[[@pntd.0008148.ref038]\]. After the above steps, we built a qualified *Echinococcus* tapeworm sequence database.

Workflow construction {#sec012}
---------------------

Bioinformatics workflow was constructed to identify *Echinococcus* spp. reads with five main steps ([Fig 1](#pntd.0008148.g001){ref-type="fig"}). 1) Raw data were first processed with SOAPnuke (v1.5.6) \[[@pntd.0008148.ref039]\] and Fastp (v0.19.5) \[[@pntd.0008148.ref040]\] to remove low-quality reads. 2) Clean data were mapped to *Echinococcus* spp. sequence database with Kraken (v0.10.5) \[[@pntd.0008148.ref041]\], and the candidate *Echinococcus* spp. reads were extracted from mapping results. 3) Remove reads sourced from humans with Snap-aligner (1.0beta.23) \[[@pntd.0008148.ref042]\]. 4) Low-complexity reads were difficult to be classified accurately, thus might introduce false-positive results, and were removed with PRINSEQ (v0.20.4) \[[@pntd.0008148.ref043]\]. 5) Remove reads of other taxa. To remove reads of other microorganisms (such as bacteria, fungus, and viruses) either from plasma or introduced by the experimental process, left candidate reads were separately mapped to the *Echinococcus* database and comprehensive database (NCBI nt) by BLASTn \[[@pntd.0008148.ref037]\]. Reads with poor mapping results (identity \< 97%, coverage \< 92%, and e-value \> 1e-5) to the *Echinococcus* spp. sequences would be removed and reads that had a better mapping result to other species would also be removed.

![Reference database construction and analysis workflow.\
Construction of *Echinococcus* spp. reference sequence database (left). Analysis workflow of cell-free *Echinococcus* spp. DNA reads identification (right).](pntd.0008148.g001){#pntd.0008148.g001}

Workflow evaluation {#sec013}
-------------------

To evaluate the reliability of the workflow, we tested it with three datasets, including simulated data, cell lines deep sequencing data, and cfDNA sequencing data of individuals from non-endemic areas. Simulated data (paired-end 100bp) were produced by wgsim (<https://github.com/lh3/wgsim>) with human reference and *Echinococcus* spp. sequences. Data of cell lines produced with the same sequencing platform were used as a negative control. CfDNA sequencing data of 107 pregnant women from an ongoing study living in non-endemic areas were also used as a negative control. All three datasets were analyzed with the workflow to evaluate its performance.

Annotation and fragment length calculation {#sec014}
------------------------------------------

Identified cell-free *Echinococcus* spp. DNA reads were annotated with information from *Echinococcus* spp. sequence database. The annotation consisted of the release source and the species annotation. The read pairs were labeled according to their best mapping results to the mitochondrial or nuclear sequences which indicated their release source. The species annotation of the sample was determined similarly as the species with the most reads labeled. Based on the samples' species annotation results, *E*. *granulosus* and *E*. *multilocularis* \[[@pntd.0008148.ref044]\] were chosen as reference for the cell-free *Echinococcus* spp. DNA features exploration. Since a more complete mitochondrial genome of *E*. *granulosus* has been published \[[@pntd.0008148.ref045]\], we replaced the mitochondrial sequence of Tsai, *et al*. \[[@pntd.0008148.ref044]\] with the most updated one. Read pairs from *E*. *granulosus* annotated samples and *E*. *multilocularis* annotated samples were pooled separately and remapped with BWA (v0.7.16) \[[@pntd.0008148.ref046]\] to their corresponding references to get the mapping positions and fragment length. Based on the mapping results, the insert size was calculated with Picard (<http://broadinstitute.github.io/picard>). Fragment length distribution figures were produced with R version 3.3.2 (<https://www.R-project.org/>). Visualization of mapping positions of cell-free *Echinococcus* spp. DNA reads was achieved with Circos \[[@pntd.0008148.ref047]\].

Analysis of sequencing data volume and positive detection {#sec015}
---------------------------------------------------------

Based on *Echinococcus* spp. reads proportion, plasma cfDNA concentration, and statistical model, we analyzed the relationship between the amount of sequencing data and positive detection. Sequencing of cfDNA and cell-free *Echinococcus* spp. DNA detection can be regarded as a random sampling process. According to the hypergeometric distribution formula ([1](#pntd.0008148.e001){ref-type="disp-formula"}), where population size (*N*) = total number of cfDNA fragments, overall target number (*M*) = total number of cell-free *Echinococcus* spp. DNA. The number of draws (*n*) = sequencing reads amount, and the number of observed success (*x*) = detected cell-free *Echinococcus* spp. reads counts. Based on the concentration of cfDNA in the plasma of each sample, we converted the total quality of cfDNA contained in 1ml plasma to base pairs (bp) according to the formula 1pg = 978Mb \[[@pntd.0008148.ref048]\]. According to the literature, the average length of cfDNA is 170 bp \[[@pntd.0008148.ref049]\], and then we estimated total cfDNA fragment counts (*N*) of 1ml plasma. The total number of cell-free *Echinococcus* DNA (*M*) present in 1 ml plasma was estimated based on their proportion detected by sequencing. Then based on the formula ([2](#pntd.0008148.e002){ref-type="disp-formula"}), we can calculate the probability to get at least one cell-free *Echinococcus* spp. reads detection at a certain amount of sequencing data ([S4 Table](#pntd.0008148.s004){ref-type="supplementary-material"}).

![](pntd.0008148.e001.jpg){#pntd.0008148.e001g}

P

(

X

=

x

)

=

(

M

x

)

(

N

−

M

n

−

x

)

(

N

n

)

![](pntd.0008148.e002.jpg){#pntd.0008148.e002g}

P

(

X

≥

1

)

=

1

−

P

(

X

=

0

)

Results {#sec016}
=======

Samples collection and sequencing data production {#sec017}
-------------------------------------------------

Blood samples were collected from 23 echinococcosis patients. The average age of these patients (10 males and 13 females) was 38 years ([Table 1](#pntd.0008148.t001){ref-type="table"}). Plasma cfDNA was sequenced with the BGISEQ-500 platform and produced a total of 6,480,520,054 paired-end reads with the amount of data about 1.30 Tb. After quality control, an average of 235,025,384 paired-end clean reads per sample were left.

Performance evaluation of the analysis workflow {#sec018}
-----------------------------------------------

Simulated data, cell lines sequencing data and control human data were used to evaluate the workflow. The simulated data set included 300,000,000 paired-end reads from humans, 1,000 paired-end reads from *Echinococcus* spp. nuclear genome and 100 paired-end reads from *Echinococcus* spp. mitochondrial genome ([S1 Table](#pntd.0008148.s001){ref-type="supplementary-material"}). After analysis with the workflow, 99.5% of the *Echinococcus* spp. nuclear genome reads were identified, 98.0% of the *Echinococcus* spp. mitochondrial genome reads were identified, and no human reads were wrongly identified ([S1 Table](#pntd.0008148.s001){ref-type="supplementary-material"}).

As for the negative controls, DNA of cell lines was sequenced and 2,047,723,953 paired-end clean reads were produced. Evaluation of the cell lines data with the workflow showed that no *Echinococcus* spp. reads were detected. Besides, control data from 107 individuals with a total of 6,838,155,312 paired-end clean reads were used to evaluate the workflow and *Echinococcus* spp. reads were not detected from these data.

Detection of *Echinococcus* spp. infection {#sec019}
------------------------------------------

We used cfDNA sequencing and ELISA test to compare their performance in *Echinococcus* spp. infection detection with plasma samples from echinococcosis patients. Sequencing data of plasma cfDNA were analyzed with the analysis workflow. Cell-free *Echinococcus* spp. DNA reads were identified from all the sequencing data (23/23), with an average of 565 read pairs per sample ([Table 1](#pntd.0008148.t001){ref-type="table"}). To determine the *Echinococcus* species from cfDNA sequencing data, *Echinococcus* spp. reads were classified with taxonomic information. Species classification results of the identified *Echinococcus* spp. reads showed that 22 samples had most reads annotated to *E*. *granulosus*, and the remaining sample (S1) had most reads annotated to *E*. *multilocularis* ([S3 Table](#pntd.0008148.s003){ref-type="supplementary-material"}). In comparison, the ELISA IgG kit identified 17 (73.9%) of the plasma samples of patients (N = 23) as positive. To be specific, out of the 14 surgically confirmed patients, 12 (85.7%) were positive. Out of the 9 non-surgery patients, 5 (55.6%) were positive ([Table 1](#pntd.0008148.t001){ref-type="table"}).

Lesion samples from surgery (n = 14) were tested with PCR methods \[[@pntd.0008148.ref036]\] to validate the infection status and identify parasite species. All the 14 lesion samples were PCR positive ([Table 1](#pntd.0008148.t001){ref-type="table"}) which confirmed *Echinococcus* spp. tapeworm infection of these patients. According to PCR species differentiation results, 13 lesion samples were identified as *E*. *granulosus* infection and one lesion sample (S1) as *E*. *multilocularis* infection. The patient corresponding to S1 should be an AE patient, and other patients were confirmed as CE patients. Species identification results of PCR consisted of sequencing data analysis, which validated the plasma cfDNA sequencing methods.

Quantification of cell-free *Echinococcus* spp. DNA in plasma samples {#sec020}
---------------------------------------------------------------------

To quantify cell-free *Echinococcus* spp. DNA in plasma, we calculated cell-free *Echinococcus* spp. DNA reads proportion in total clean reads of each sample, and the proportion ranged from 1.8e-5 to 4.0e-9 ([Fig 2](#pntd.0008148.g002){ref-type="fig"}). Given the very low proportion of cell-free *Echinococcus* spp. DNA reads in the sequencing data, we normalized the identified *Echinococcus* spp. reads to total sequencing data with Read-Pairs Per Million (RPM) in order to facilitate comparison between samples. RPM was defined as *Echinococcus* spp. read counts per million sequencing data from one sample. Mean and median RPM of 22 CE patients were 0.433 and 0.082 respectively (ranging from 0.004 to 2.530) ([Table 1](#pntd.0008148.t001){ref-type="table"}), and the RPM of the only one AE sample was 18.096.

![Cell-free *Echinococcus* spp. DNA reads proportion in total clean reads of the corresponding sample.\
A scatter plot shows the detected cell-free *Echinococcus* spp. read pairs proportion (log~10~) to all clean sequencing read pairs in each sample. The dashed line represents the mean value of 22 *E*. *granulosus* samples, and the solid line represents their median value. The results showed that the overall concentration of cell-free *Echinococcus* spp. DNA in plasma was at a low level.](pntd.0008148.g002){#pntd.0008148.g002}

Based on the *Echinococcus* spp. DNA reads proportion, we calculated the probability to get at least one cell-free *Echinococcus* spp. read detection at different amounts of sequencing data ([S4 Table](#pntd.0008148.s004){ref-type="supplementary-material"}). The results showed that sequencing with 50 million reads would make 72.73% (16/22) of CE samples with over 80% probability to get positive results, sequencing with 200 million reads would make 90.91% (20/22) of CE samples with over 80% probability to get positive results, and sequencing with 400 million reads would make all 22 CE samples with over 80% probability to get positive results.

Release source of cell-free *Echinococcus* spp. DNA {#sec021}
---------------------------------------------------

By reads mapping to the reference genomes, we traced cell-free *Echinococcus* spp. DNA to their genome release source. The analysis showed that most reads were from the nuclear genome, and only a small proportion was released from the mitochondrial genome. A small amount of mitochondrial sourced reads was identified in only 7 CE samples (7/22) and the average proportion was 2.08% (ranging from 0.74% to 7.69%) ([S2 Table](#pntd.0008148.s002){ref-type="supplementary-material"}). To calculate reads per genome size, we normalized the reads counts by the genome size of nuclear and mitochondrial ([S2 Table](#pntd.0008148.s002){ref-type="supplementary-material"}). For the seven CE samples detected with mitochondrial reads, reads per genome size of mitochondrial were from 5.66e-5 to 3.39e-4, and reads per genome size of nuclear were from 1.05e-7 to 4.68e-6. The reads per genome size value of mitochondria are all higher than that of nuclear, and the value of mitochondria was between 48.35 and 539.96 (median 75.78) times that of nuclear in the seven CE samples. For the AE sample, the mitochondrial sourced reads proportion was 0.19%, and reads per genome size of mitochondrial and nuclear were 1.54e-3 and 9.67e-5.

Based on the reads mapping, we further traced the release positions of cell-free *Echinococcus* spp. DNA from the genome. Given the low proportion of cell-free *Echinococcus* spp. DNA reads from the 22 CE samples, we pooled their reads and got a total of 1,852 read pairs. The number of cell-free *Echinococcus* spp. DNA read pairs of the AE sample was 11,140. These reads were mapped to the reference genomes of *E*. *granulosus* and *E*. *multilocularis* separately. The reads coverage of *E*. *granulosus* was 213,587 bp, which accounted for about 0.19% of the whole reference genome. The reads coverage of *E*. *multilocularis* was 1,232,072 bp, which accounted for about 1.07% of the whole reference genome. Mapping positions of cell-free *Echinococcus* spp. DNA reads showed that they appeared to be evenly distributed across the genomes ([Fig 3](#pntd.0008148.g003){ref-type="fig"}, [S1](#pntd.0008148.s006){ref-type="supplementary-material"}--[S5](#pntd.0008148.s010){ref-type="supplementary-material"} Figs)

![The distribution of cell-free *E*. *granulosus* DNA reads on the nuclear genome.\
The circulation genome visualization showed the *E*. *granulosus* reads mapping position on the nuclear genome (outermost blue circle). Eighteen scaffolds longer than 1Mb were displayed in the separate fragments (Scf1-Scf18). Scaffolds shorter than 1Mb were concatenated to display (Scfshort1M). The inner orange circle represents the count of patients with reads detected in the region. Circle figures of the *E*. *granulosus* mitochondrial genome were put in the supplementary materials ([S1 Fig](#pntd.0008148.s006){ref-type="supplementary-material"}).](pntd.0008148.g003){#pntd.0008148.g003}

To analyze the distribution of identified reads in the genome, we calculated the coverage of these reads. Total coverage of 22 CE samples' reads were 213,587 bp. To analyze the overrepresented regions, we calculated the sample counts of the mapped regions. Most of the regions (211,342 bp, 98.95%) were covered with only one sample, less than 1% mapped length (1,983 bp, 0.93%) were covered with two samples, and very small region (262 bp, 0.12%) were covered with three samples. In order to intuitively compare the coverage between different samples, we plot the coverage of the samples detected with more than 100 read pairs ([S2 Fig](#pntd.0008148.s007){ref-type="supplementary-material"} and [S3 Fig](#pntd.0008148.s008){ref-type="supplementary-material"}).

Fragment length of cell-free *Echinococcus* spp. DNA {#sec022}
----------------------------------------------------

According to reads mapping to *Echinococcus* spp. genome references, fragment length of cell-free *Echinococcus* spp. DNA was inferred from the insert size of the read pairs. While human cfDNA showed an obvious peak at around 166 bp, cell-free *Echinococcus* spp. DNA fragment length distribution showed a different pattern. Cell-free *Echinococcus* spp. DNA fragment of CE showed a longer length range without an obvious peak ([Fig 4](#pntd.0008148.g004){ref-type="fig"}). Cell-free *Echinococcus* spp. DNA fragment of the AE sample showed a more regular distribution pattern with an obvious peak at about 135 bp, which was shorter than human cfDNA ([S6 Fig](#pntd.0008148.s011){ref-type="supplementary-material"}).

![Fragment length distribution of cell-free *E*. *granulosus* DNA.\
The fragment length of cfDNA was calculated by the insert size of read pairs. The fragment length of cell-free *E*. *granulosus* DNA had a broad range than human cfDNA.](pntd.0008148.g004){#pntd.0008148.g004}

Discussion {#sec023}
==========

With ultra-high-throughput sequencing technology, using plasma samples from clinically diagnosed echinococcosis patients, we identified the existence of cell-free *Echinococcus spp*. DNA in plasma, quantified the amount per sample, confirmed its low concentration and described its characteristics. The results revealed that high-throughput sequencing of plasma cfDNA could serve as a useful tool for cell-free *Echinococcus* spp. DNA studies and improve our understanding of *Echinococcus* spp. infection in the human body. Plasma cfDNA has shown its usefulness in NIPT \[[@pntd.0008148.ref023]\], tumor monitoring \[[@pntd.0008148.ref024]\], and pathogens detection \[[@pntd.0008148.ref025]\]. Several attempts were made using cfDNA in *Echinococcus* spp. detection from plasma or serum with PCR-based methods, but their overall sensitivity was only 20% to 25% \[[@pntd.0008148.ref031]--[@pntd.0008148.ref033]\]. The low sensitivity could be due to non-existence, or low concentration of cfDNA of the parasite in the circulation, which showed our limited understanding of the cfDNA of *Echinococcus spp*. tapeworms. As one of the most neglected tropical diseases and zoonosis, echinococcosis poses serious public health threats to endemic areas. Given the increase of global trade, tourism, and immigration, people of non-endemic regions could also be diagnosed with echinococcosis \[[@pntd.0008148.ref050]--[@pntd.0008148.ref052]\]. Effective detection and diagnosis methods are the premises of controlling echinococcosis, and cfDNA could be a promising tool for clinical diagnosis. We are the first using high-throughput sequencing technology to evaluate the existence, quantity, and characteristics of cell-free *Echinococcus* spp. DNA in plasma of echinococcosis patients.

The existence of *E*. *granulosus* DNA in the blood circulation of the echinococcosis patients was questioned by Chaya *et al*. who believed that the cfDNA of the parasite would only enter the blood circulation when the hydatid cyst(s) ruptured \[[@pntd.0008148.ref031]\]. Baraquin *et al*. confirmed the existence of cfDNA of *E*. *multilocularis* in AE patients and used the very low concentration of cfDNA to explain the low sensitivity of their PCR test \[[@pntd.0008148.ref032]\]. Low concentration of target DNA in plasma is a common situation for cfDNA studies. Cell-free fetal DNA in maternal plasma cfDNA accounts for about 10% to 15% \[[@pntd.0008148.ref053],[@pntd.0008148.ref054]\], and circulating-tumor DNA comprises about 0.01% to 10% or more in cancer patients plasma cfDNA \[[@pntd.0008148.ref055],[@pntd.0008148.ref056]\]. Based on high-throughput sequencing data and bioinformatics workflow, we identified the cell-free *Echinococcus* spp. DNA reads from sequencing data of all the samples. Compared with cell-free fetal DNA and circulating-tumor DNA, cell-free *Echinococcus* spp. DNA in plasma is extremely low, whose proportion ranged 1.8e-5 to 4.0e-9 in these samples ([Fig 2](#pntd.0008148.g002){ref-type="fig"}). Indeed, this low concentration may explain the low sensitivity of the PCR-based methods \[[@pntd.0008148.ref031]--[@pntd.0008148.ref033]\]. Besides the low concentration, we identified the difference between the cell-free *Echinococcus* spp. DNA from CE and AE samples. The AE sample had much more cfDNA identified than the CE samples, which could be due to the different developmental mechanisms of metacestode in the human body. Compared with *E*. *granulosus*, the metacestode of *E*. *multilocularis* is an infiltrating lesion composed of aggregated microvesicles, necrosis cells, and fibrosis cells, which have no clear edge to the host tissues \[[@pntd.0008148.ref001]\] and relatively high concentration of cell-free *Echinococcus* spp. DNA in the AE sample could be due to the mixture of necrotic parasite tissue and actively proliferating tissues. This is in line with the previous finding that the sensitivity of PCR-based methods in AE samples was higher than in CE samples \[[@pntd.0008148.ref031]--[@pntd.0008148.ref033]\]. As we only collected one AE sample, it needed further verification with more samples. Plasma samples were also tested with ELISA assays to detect the antibody, and the positive results were found in 16 out of 22 CE patients ([Table 1](#pntd.0008148.t001){ref-type="table"}). Serological tests may be influenced by lots of factors, and difficult to standardize \[[@pntd.0008148.ref018]--[@pntd.0008148.ref020]\]. In contrast, DNA detection is a more direct and objective biomarker.

Low concentration is the major challenge to apply cell-free *Echinococcus* spp. DNA testing in routine clinical settings. To estimate the minimal number of reads needed to get cell-free *Echinococcus* spp. DNA, we treated sequencing as a random sampling process, and the number of sequencing reads regarded as sampling times. We estimated total cfDNA fragment counts and *Echinococcus* spp. fragment counts according to cfDNA concentration and existed detection results. By using hypergeometric distribution, we calculated the probability of each sample to get cell-free *Echinococcus* spp. DNA. detection with different sequencing amounts. We found that sequencing with 50 million reads would make 72.73% (16/22) of CE samples with over 80% probability to get positive results, while sequencing with 400 million reads would make all 22 CE samples identified with over 80% probability to get positive results ([S4 Table](#pntd.0008148.s004){ref-type="supplementary-material"}). The lower the concentration, the harder it is to be detected, and increasing the amount of sequencing can increase the chance of positive detection. The concentration may vary greatly between individuals. Just like the cell-free DNA of fetus in maternal plasma, which are influenced by gestational age, maternal BMI, fetal aneuploidy status and other factors \[[@pntd.0008148.ref057]\]. The concentration of cell-free *Echinococcus* spp. DNA might also be affected by many factors, such as disease status, parasite species, lesion size, and position, which need more comprehensive samples to explore its association with different patterns.

Cell-free DNA *Echinococcus* spp. DNA in plasma could not only detect the etiology of the patients' infection but also facilitate the species identification. Traditional species identification of *Echinococcus* spp. in echinococcosis patients is always invasive, which relies on the product of surgery or puncture. Surgery is only recommended for part of echinococcosis patients, puncture can assist to get specimens for confirming etiology. However, while puncture is of high diagnostic value and safe in most AE patients \[[@pntd.0008148.ref058]\], it is not recommended for some CE patients, especially for CE4, CE5 and lung cysts, which may pose the risks of allergic reactions and anaphylaxis \[[@pntd.0008148.ref001],[@pntd.0008148.ref008]\]. In this study, species annotation of cell-free *Echinococcus* spp. DNA was analyzed according to reads mapping results. Given the genome sequence similarity between *Echinococcus* species and limited reference sequences available, part of cell-free *Echinococcus* spp. DNA reads may be classified into closely related species of genus *Echinococcus*, but the majority of the reads should be classified correctly. Consistency of species classification between cell-free *Echinococcus* spp. DNA and lesion samples' PCR results proved their accuracy in species annotation. This cfDNA sequencing-based taxonomy annotation method may provide an innovative non-invasive alternative to obtain more detailed etiology information. Species identification of echinococcosis patients could provide more valuable information for guiding clinical management and research such as molecular epidemiology \[[@pntd.0008148.ref059]\].

Tracing cell-free *Echinococcus* spp. DNA release sources could provide more background information. Based on cell-free *Echinococcus* spp. DNA reads mapping, we further analyzed their genome release source. Sequence origin analysis showed that much more cell-free *Echinococcus* spp. DNA was released from the nuclear genome than the mitochondrial genome. This phenomenon may be due to the fact that the genome size of nuclear is much larger than mitochondria. The overall low proportion of mitochondrial-derived cell-free *Echinococcus* spp. DNA in plasma may also partially explain the low positive rate of mitochondrial gene based PCR \[[@pntd.0008148.ref031]--[@pntd.0008148.ref033]\]. However, reads per genome size of mitochondria were about 75.78 times larger than that of nuclear, which could be due to the multi copies of mitochondria \[[@pntd.0008148.ref044]\]. The position distribution of cell-free *Echinococcus* spp. DNA on the genome were analyzed with reads mapping to *E*. *granulosus* and *E*. *multilocularis* genome references. We found that the release positions of cell-free *Echinococcus* spp. DNA were nearly evenly distributed on the genome. It looks like there are some hotspots of cell-free *Echinococcus* spp. DNA release on the genome, but these spots are more gathered on the short and not well-assembled regions of the available genome references. With higher quality references in the future, the distribution of cell-free *Echinococcus* spp. DNA on the genome could be more evenly distributed.

Size characteristics of cfDNA is an important biological property \[[@pntd.0008148.ref035]\]. To have a deep understanding of cell-free *Echinococcus* spp. DNA, we analyzed its fragment size with sequencing data. Literature shows that cfDNA could have different size pattern according to research settings \[[@pntd.0008148.ref035]\]. Fetal cfDNA in maternal plasma has a shorter fragment size distribution compared with maternal cfDNA \[[@pntd.0008148.ref060]\]. In certain types of cancer patients, tumor sourced cfDNA is concentrated in short fragments \[[@pntd.0008148.ref061]\]. Fragment size analysis of cell-free *Echinococcus* spp. DNA in our study showed that they had a different length distribution to human-sourced cfDNA. We found that cell-free *Echinococcus* spp. DNA of CE had a broad length range ([Fig 4](#pntd.0008148.g004){ref-type="fig"}), but that of AE had an obvious peak at about 135 bp ([S6 Fig](#pntd.0008148.s011){ref-type="supplementary-material"}). The size profile of cfDNA is relevant to their release mechanism such as apoptosis, necrosis and actively release \[[@pntd.0008148.ref062],[@pntd.0008148.ref063]\]. Quite different fragment size features of cell-free *Echinococcus* spp. DNA in CE and AE could be related to their developmental mechanism of metacestode in the human body. Tumor like AE lesions may give some explanation to its overall short fragment length, and similar phenomenon of tumor-derived DNA in plasma of hepatocellular carcinoma patients was also observed \[[@pntd.0008148.ref064]\]. As there was only one accidental AE sample, this phenomenon needs more research to validate. Though the exact release mechanism of cfDNA is still unclear, it doesn't affect the application of size properties in diagnostics \[[@pntd.0008148.ref035]\]. As for cell-free *Echinococcus* spp. DNA, their fragment size features may facilitate their detection in future studies.

The cfDNA sequencing-based method relies on high quality and comprehensive database, but existing genome references of *Echinococcus* spp. are limited, and only several genome references are available \[[@pntd.0008148.ref044],[@pntd.0008148.ref065],[@pntd.0008148.ref066]\] whose quality is far from perfect. More importantly, sequence contamination is a serious problem for cell-free *Echinococcus* spp. DNA detection and contaminated sequence database might introduce false-positive results. Since the *Echinococcus* spp. tapeworm samples are always separated from host tissue \[[@pntd.0008148.ref044],[@pntd.0008148.ref065],[@pntd.0008148.ref066]\], it is not easy to remove the contamination of host thoroughly by experimental processing. In the process of genome constructing, some host sequences may mix into the parasite sequence, which is a common problem for genomes construction \[[@pntd.0008148.ref067]\]. It is essential to qualify the genome sequence with bioinformatics methods after downloading from the public database, instead of using it directly \[[@pntd.0008148.ref067]\]. In our study, we filtered the *Echinococcus* spp. sequence database with their common host genomes such as sheep, humans, and mice, and evaluated the workflow with simulation data, cell line data, and negative control data, which all showed that qualified database introduced no false-positive results.

High-throughput sequencing facilitated identifying, quantifying and analyzing the characteristics of cell-free *Echinococcus* spp. DNA in human plasma. These comprehensive characteristics could help the application of cell-free *Echinococcus* spp. DNA in the future diagnosis of echinococcosis. However, for the very low concentration of cell-free *Echinococcus* spp. DNA, their even distribution on the genome, and the high sequencing depth and cost, the method requires further optimization. To increase the application of cell-free *Echinococcus* spp. DNA, we could think of some areas to be explored in the future study, for example, capturing cell-free *Echinococcus* spp. DNA with probes covered the whole genome and enriching the concentration of cell-free *Echinococcus* spp. DNA by host sequence removal. As for clinical application scenarios, massive sequencing of plasma cfDNA to detect cell-free *Echinococcus* spp. DNA may not be suitable for routine clinical examination yet, but it could be used for differential diagnosis, in which existing clinically methods cannot give clear conclusions. For example, the CL patients can be further diagnosed with plasma cfDNA sequencing and avoid the risk of invasive diagnosis.

Supporting information {#sec024}
======================

###### Evaluation of analysis workflow with simulation data.

Simulation data showed that no human reads appeared in the results, and most *Echinococcus* spp. reads were identified by the analysis workflow. Counts in the table were read pairs.

(XLSX)

######

Click here for additional data file.

###### Release source of identified *Echinococcus* spp. reads.

Most of the identified *Echinococcus* spp. reads were released from the nuclear genome. Only eight samples were identified with mitochondrial reads.

(XLSX)

######

Click here for additional data file.

###### Species classification with cfDNA reads mapping.

The table showed the species classification results from each sample with cfDNA sequencing read pairs. The sample was classified to the species with most read pairs mapping.

(XLSX)

######

Click here for additional data file.

###### Amount of sequencing data and reads detection.

Statistical analysis with hypergeometric distribution to estimate the probability to get positive results with different sequencing amount.

(XLSX)

######

Click here for additional data file.

###### Plasma volume and DNA extraction methods.

The volume of plasma and kit used for each sample.

(XLSX)

######

Click here for additional data file.

###### Circle figure of *E. granulosus* samples based on the mitochondrial genome.

The circulation genome visualization showed the *E*. *granulosus* reads mapping position (outermost blue circle). The inner orange circle represents the count of patients with reads detected in the region.

(TIFF)

######

Click here for additional data file.

###### Circle figure of multiple *E. granulosus* samples based on the nuclear genome.

Seven *E*. *granulosus* samples detected with more than 100 *Echinococcus* spp. read pairs were displayed based on the nuclear genome. Green and red circles indicate different samples.

(TIF)

######

Click here for additional data file.

###### Circle figure of multiple *E. granulosus* samples based on the mitochondrial genome.

Seven *E*. *granulosus* samples detected with more than 100 *Echinococcus* spp. read pairs were displayed based on the mitochondrial genome. Green and red circles indicate different samples.

(TIF)

######

Click here for additional data file.

###### Circle figure of the *E. multilocularis* sample based on the nuclear genome.

The circulation genome visualization showed the *E*. *multilocularis* reads mapping position (outermost blue circle). Ten scaffolds longer than 1Mb were displayed in the separate fragment (Scf1-Scf10). Scaffolds shorter than 1Mb were concatenated to display (Scfshort1M).

(TIF)

######

Click here for additional data file.

###### Circle figure of the *E. multilocularis* sample based on the mitochondrial genome.

The circulation genome visualization showed the *E*. *multilocularis* reads mapping position (outermost blue circle).

(TIF)

######

Click here for additional data file.

###### Fragment length distribution of the *E. multilocularis* sample.

Overall fragment length distribution of *E*. *multilocularis* cfDNA was shorter than that of humans.

(TIFF)

######

Click here for additional data file.

The authors would like to thank China National GeneBank for sequencing the samples of this study. We would also like to thank Yue Zhao and Ying Yi for their previous analysis work on the project.

10.1371/journal.pntd.0008148.r001

Decision Letter 0

Casulli

Adriano

Deputy Editor

Koziol

Uriel

Guest Editor

© 2020 Casulli, Koziol

2020

Casulli, Koziol

This is an open access article distributed under the terms of the

Creative Commons Attribution License

, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

26 Nov 2019

Dear Mr Ji:

Thank you very much for submitting your manuscript \"Comprehensive characterization of cell-free Echinococcus spp. DNA in echinococcosis patients' plasma using extremely high throughput sequencing.\" (\#PNTD-D-19-01705) for review by PLOS Neglected Tropical Diseases. Your manuscript was fully evaluated at the editorial level and by independent peer reviewers. The reviewers appreciated the attention to an important problem, but raised some substantial concerns about the manuscript as it currently stands. These issues must be addressed before we would be willing to consider a revised version of your study. We cannot, of course, promise publication at that time.

We therefore ask you to modify the manuscript according to the review recommendations before we can consider your manuscript for acceptance. Your revisions should address the specific points made by each reviewer.

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Adriano Casulli

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\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*

All reviewers were generally positive regarding the manuscript and have stated that it represents a valuable contribution to the field. However, all of the reviewers expressed concerns regarding specific aspects of the manuscript and the experimental work. In particular, one main issue that two reviewers found was that only one case of E. multilocularis was investigated, and recommend either incorporating additional cases or removing this case from the manuscript (and from the title). In any case, this aspect needs to be addressed appropriately in the discussion. All reviewers also consider that the discussion should include a guideline on how cfDNA could (our could not) be used clinically in the near future as a method for diagnostics. Additional concerns should also be addressed, particularly regarding the negative controls, which should be described and presented in far greater detail. Finally, make sure to include the permit number for ethical approval of this study in the ethics statement, and to make the original data for the negative controls available as well.

Reviewer\'s Responses to Questions

**Key Review Criteria Required for Acceptance?**

As you describe the new analyses required for acceptance, please consider the following:

**Methods**

-Are the objectives of the study clearly articulated with a clear testable hypothesis stated?

-Is the study design appropriate to address the stated objectives?

-Is the population clearly described and appropriate for the hypothesis being tested?

-Is the sample size sufficient to ensure adequate power to address the hypothesis being tested?

-Were correct statistical analysis used to support conclusions?

-Are there concerns about ethical or regulatory requirements being met?

Reviewer \#1: see below

Reviewer \#2: The objectives are clearly stated. The study design is appropriate but there are some negative control results that should be presented and discussed (these dataset is mention but missing in the text). The sample size is ok. No concerns about ethical or regulatory requirements.

Reviewer \#3: The objectives of this paper were clearly described and study design is appropriate

As it could be considered as a pilot/exploratory study, number of included patient is correct

My main concerns with this study are the following:

\- among 23 patients, they had 22 CE and only one AE patient. They were included based on US examination. To my knowledge, US patterns of AE and CE are mostly different, except some cases (CL stage). In 22 out of their 23 cases, diagnosis of CE was known before biological diagnosis (molecular study)

As diagnosis of type of echinococcosis is not the key point of results but a key point of methods, i suggest that this information should be given in methods and not in results section

\- the authors used surprisingly two different strategies for DNA extraction : why ? which sample among the 23 was processed with alternative approach ?

If they processed the AE blood sample with another ADN extraction approach than that used for CE blood sample, it affect significantly all the results obtained in this study. This information is not given in the paper

\- preanalytical conditions are crucial for cfDNA : the authors should described which was the delay observed between blood sampling and freezing, and centrifugation conditions (one step ? two step ? temperature of centrifugation ? speed of centrifugation g ?)

\- at least, please indicate if all patients were included at diagnosis and systematically sampled before surgical and/or medical treatment and/or after beginning of treatment

\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\--

**Results**

-Does the analysis presented match the analysis plan?

-Are the results clearly and completely presented?

-Are the figures (Tables, Images) of sufficient quality for clarity?

Reviewer \#1: see below

Reviewer \#2: Some negative control results are mention but need to be presented and discussed. The figures 3 and 4 need to be improved. Some statistical analysis/simulations in relation to the number of reads generated (more than 80 millions) and the number of reads detected may give some more support to presented results.

Reviewer \#3: This study give interesting but very contrasting results between AE and CE patients, especially cfDNA yield and structure observed between the two species of Echinococcus

Except my previous limitations given previously, results were clearly expressed

\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\--

**Conclusions**

-Are the conclusions supported by the data presented?

-Are the limitations of analysis clearly described?

-Do the authors discuss how these data can be helpful to advance our understanding of the topic under study?

-Is public health relevance addressed?

Reviewer \#1: see below

Reviewer \#2: The identification of Echinoccoccus spp. cell free DNA reads is really marginal in some patients, however the authors conclude that the technique es valid in 100% cases. This issue should be discussed and mention in conclusions.

Reviewer \#3: I disagree with author\'s statement (Lines 419-421): if puncture is prohibited for CE patients, it is of high diagnostic value and safe in AE patients. Please see

\- Review in Clin Microbiol Review 2019 (Wen, Vuitton\... your ref number one)

\- Efficacy of ultrasound-guided core-needle biopsy in the diagnosis of hepatic alveolar echinococcosis: a retrospective analysis. Bulakci et al. Parasite 2016;23:19 (to be included in your references)

Include reference of crucial usefulness of imaging technique in diagnosis of echinococcosis

Last sentence of the paper is very interesting: \"given the high sequencing depth and cost, the application scenarios and potential need to be further explored\". Please develop what you mean

Please balance more your results (even if very interesting contrast) : you only included one AE patient !! (Lines 411-412)

\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\--

**Editorial and Data Presentation Modifications?**

Use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity. If the only modifications needed are minor and/or editorial, you may wish to recommend "Minor Revision" or "Accept".

Reviewer \#1: see below

Reviewer \#2: (No Response)

Reviewer \#3: Introduction

Please include some sentences about very young cysts, called CL (please refer to review of Wen, Vuitton and to Engler et al. Simple liver cysts and cystoid lesions in hepatic alveolar echinococcosis: a retrospective cohort study with Hounsfield analysis. Parasite 2019;26 :54)

Line 396 : Baraquin (instead of Baraquin)

\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\--

**Summary and General Comments**

Use this section to provide overall comments, discuss strengths/weaknesses of the study, novelty, significance, general execution and scholarship. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. If requesting major revision, please articulate the new experiments that are needed.

Reviewer \#1: Ji et al. report on work concerning the detection of cell-free parasite DNA in serum samples of echinococcosis patients by Next Generation Sequencing approaches. The authors identified parasite DNA in 23 samples (of 23 different patients). They demonstrated that parasite cfDNA concentrations are very low in echinococcosis patients, which could explain why several PCR approaches towards this aim so far failed (or only showed very low sensitivity). To my knowledge, successful detection of cfDNA in echinococcosis patients by high throughput NGS has so far not been published which is, indeed, a strong point of this work. I agree with the authors that basic knowledge on cfDNA realeased by these parasites into host serum is important for future development of respective molecular diagnostics methodology (although, of course, not feasible at the moment due to high costs). In general the work is well written and the NGS methods seem to have been carried out in a professional way (although a bioinformatics expert should closer evaluate this part of the work). There are, however, also several limitations in this work. On the one hand, the authors do not provide any suggestions on how, based on their data, proper molecular diagnostics of serum samples can be carried out in the future (their data rather suggest that this will not be possible for long time -- which, of course, would be a valid result if properly discussed). The most serious limitation I see is that only one E. multilocularis sample was analyzed. This is surely not enough to come to significant conclusions, particularly concerning the general suitability of echinococcosis serum samples for molcular diagnostics (in E. multilocularis the cfDNA concentration was much higher), or concerning differential diagnostic methods that can distinguish between E. granulosus and E. multilcularis. On the one hand, this could be solved by taking out the E. multilocularis sample and by concentrating only on E. granulosus (stating, in the end, that it is almost impossible to detect E. granulosus DNA in plasma samples). On the other hand, I would consider a significant step forward (i.e. ‚importance' as outlined in the PNTD guidelines for publication) in echinococcosis diagnostics the comprehensive analysis of both E. multilocularis and E. granulosus cfDNA in patient plasma. I thus strongly suggest that additional E. multilocularis samples are analyzed so that the title of the study (Comprehensive characterization of cf Echinococcus spp. DNA....) is justified.

Additional points

1\) The English of the manuscript is generally quite well and comprehensible. Nevertheless, another round of checking readability would be good (particularly proper use of specifying articles like ‚the' and ‚a' as well as correct use of plural/singular).

2\) L146-148: why is it likely that parasite DNA can be found in plasma because of complex host-parasite interaction? This kind of interaction could exclusively be mediated by proteins.

3\) Shouldn't there be a permit no. for the ethics statement?

4\) Paragraph 2.2.: please be specific on when the plasma samples have been taken. I guess it was before (!) treatment. This should be pointed out in detail.

5\) Paragraph 2.5.: please be specific on when the ELISA has been carried out.

6\) Related to point 4 and 5: it would, of course, strengthen the study if cfDNA analyses (and maybe ELISA) would have been carried out also after treatment (especially chemotherapy) at least for some samples.

7\) The authors only superficially discussed why they detected so much more cfDNA in the E. multilocularis patient. I think the big difference is that E. multilocularis metacestode material in the human host is generally a mixture between nectrotic parasite tissue and actively proliferating tissue. Hence, cfDNA could result from parasite cell necrosis. This should be taken into account and could also explain why there were shorter fragments in the E. multilocularis sample.

8\) L 437/438: shouldn't it be possible to calculate reads per genome size? This would give us hints as to whether nuclear or mitochondrial genome are over-represented.

9\) Fig. 3: At least in the mitochondrial genome I see some hotspots. Which are these?

10\) Table 1: please add for all samples which species was identified according to NGS reads.

11\) References: genus/species in italics.

Reviewer \#2: Review of "Comprehensive characterization of cell-free Echinococcus spp. DNA in echinococcosis patients' plasma using extremely high throughput sequencing" by Ji and co-authors.

In the present manuscript (MS) Ji and coauthors report the results of applying extremely high throughput sequencing to detecting cell-free Echinococcus spp. DNA in plasma of echinococcosis patients.

Authors do a well-organizer presentation of auspicious results regarding the possibility of identified and partially characterize Echinococcus spp. infections from human blood samples. The MS followed a straight-forward bioinformatic pipeline to identify reads from cell free DNA from Echinococcus and more importantly to discard false positive results. This approaches has been successfully applied for others pathogens.

This MS is well written and my overall impression of it is positive, however I have some concerns that I think the authors need to address before publications. Some suggested changes could improve the robustness and presentation of the MS.

Major Comments:

1\. Negative control data are not mention (Humans with no diagnostic Echinoccoccus) until the Results sections. This is a major problem since no results about this control dataset is presented in the MS (see Line 295, pg. 8). Given that detection of Echinoccoccus spp. reads is so marginal in the whole NGS dataset, the results of the bioinformatics pipeline applied to this 107 human blood samples are key to support presented positive results. Negative controls are not included under accession number of CNGB.

2\. The number of reads identified is rather marginal; this is particularly noticeable when considering the millions of reads generated in each sequencing experiment (median 235 million reads). Authors should discuss more thoroughly these results and possible improvements that need to be done in order to make this approach clinically feasible. For instance, I wonder if single reads could improve the detection of cell free DNA. In relation to this the authors should consider do some statistical/modeling analysis to estimate the minimal number of reads generated to get positive results.

Minor Comment (not in order):

• When building the Echinococcus spp. reference database, in order to reduce

sequence contamination and get high quality genomes, authors used relatively high thresholds values (97% identity, 92% coverage, e-value) when filtering the posible sequences from host (human, mouse, etc..). Reads with high similarity to the host genomes are discarded. Authors need to clarify why these values are chosen, and as I understand the higher thresholds may allow including contaminant reads in the analysis.

• I am not quite sure if Kraken is the best option to do the "mapping". Please check or clarify.

• More analysis on the distribution of identified reads in the genome of E. spp. are needed. Are there any difference among patients? Which are the more represented regions? Is there any common characteristic in the overrepresented regions?

• Please check figures 3 and 4, both are not in resolution and are not particularly informative about genome distribution of reads.

• Clarify the meaning of CL and CE in Table 1.

Reviewer \#3: As a summary, very interesting paper and well-done study.

Authors should improve description of methods

If they processed blood from their AE patient with a distinct extraction, it is a bias. Thus, they should

\- either clearly balance their results and discussion

\- or limit their paper to their 22 CE patients (they may design another similar study focused on AE patients)

\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\--

PLOS authors have the option to publish the peer review history of their article ([what does this mean?](https://journals.plos.org/plosntds/s/editorial-and-peer-review-process#loc-peer-review-history)). If published, this will include your full peer review and any attached files.

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Reviewer \#1: No

Reviewer \#2: No

Reviewer \#3: No

10.1371/journal.pntd.0008148.r002

Author response to Decision Letter 0

14 Jan 2020

######

Submitted filename: Letter_to_editor_and_Reviewers.docx

######

Click here for additional data file.

10.1371/journal.pntd.0008148.r003

Decision Letter 1

Casulli

Adriano

Deputy Editor

Koziol

Uriel

Guest Editor

© 2020 Casulli, Koziol

2020

Casulli, Koziol

This is an open access article distributed under the terms of the

Creative Commons Attribution License

, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

18 Feb 2020

Dear Mr Ji,

We are pleased to inform you that your manuscript \'Comprehensive characterization of plasma cell-free Echinococcus spp. DNA in echinococcosis patients using extremely high-throughput sequencing.\' has been provisionally accepted for publication in PLOS Neglected Tropical Diseases.

Before your manuscript can be formally accepted you will need to complete some formatting changes, which you will receive in a follow up email. A member of our team will be in touch within two working days with a set of requests.

Please note that your manuscript will not be scheduled for publication until you have made the required changes, so a swift response is appreciated.

IMPORTANT: The editorial review process is now complete. PLOS will only permit corrections to spelling, formatting or significant scientific errors from this point onwards. Requests for major changes, or any which affect the scientific understanding of your work, will cause delays to the publication date of your manuscript.

Should you, your institution\'s press office or the journal office choose to press release your paper, you will automatically be opted out of early publication. We ask that you notify us now if you or your institution is planning to press release the article. All press must be co-ordinated with PLOS.

Thank you again for supporting Open Access publishing; we are looking forward to publishing your work in PLOS Neglected Tropical Diseases.

Best regards,

Uriel Koziol

Guest Editor

PLOS Neglected Tropical Diseases

Adriano Casulli

Deputy Editor

PLOS Neglected Tropical Diseases

\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*

The authors have succesfully reviewed their manuscript, addressing all of the main concerns raised by the reviewers.

Reviewer\'s Responses to Questions

**Key Review Criteria Required for Acceptance?**

As you describe the new analyses required for acceptance, please consider the following:

**Methods**

-Are the objectives of the study clearly articulated with a clear testable hypothesis stated?

-Is the study design appropriate to address the stated objectives?

-Is the population clearly described and appropriate for the hypothesis being tested?

-Is the sample size sufficient to ensure adequate power to address the hypothesis being tested?

-Were correct statistical analysis used to support conclusions?

-Are there concerns about ethical or regulatory requirements being met?

Reviewer \#1: To my opinion the authors have adequately addressed not only my concerns on the first version but also the concerns of all other reviewers. They have made additional analyses, provided a more detailed description of the methods, and toned down several conclusions when appropriate. The revised version of the manuscript is a well-perormed improvement of the first version and the scientific discussion in the response to reviews was thorough and inspiring. Congratulations.

Reviewer \#2: The authors take in account almost all our comments.

Reviewer \#3: (No Response)

\*\*\*\*\*\*\*\*\*\*

**Results**

-Does the analysis presented match the analysis plan?

-Are the results clearly and completely presented?

-Are the figures (Tables, Images) of sufficient quality for clarity?

Reviewer \#1: see above

Reviewer \#2: The authors modified figures 3 and 4 as suggested, did statistical analyses, clarified specific sections of the MS and response to all comments raised by reviewers.

Reviewer \#3: (No Response)

\*\*\*\*\*\*\*\*\*\*

**Conclusions**

-Are the conclusions supported by the data presented?

-Are the limitations of analysis clearly described?

-Do the authors discuss how these data can be helpful to advance our understanding of the topic under study?

-Is public health relevance addressed?

Reviewer \#1: see above

Reviewer \#2: (No Response)

Reviewer \#3: (No Response)

\*\*\*\*\*\*\*\*\*\*

**Editorial and Data Presentation Modifications?**

Use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity. If the only modifications needed are minor and/or editorial, you may wish to recommend "Minor Revision" or "Accept".

Reviewer \#1: I did not find any obvious errors.

Reviewer \#2: Accept

Reviewer \#3: (No Response)

\*\*\*\*\*\*\*\*\*\*

**Summary and General Comments**

Use this section to provide overall comments, discuss strengths/weaknesses of the study, novelty, significance, general execution and scholarship. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. If requesting major revision, please articulate the new experiments that are needed.

Reviewer \#1: see above and first review

Reviewer \#2: Authors have addressed all my comments into this new version of the manuscript. In general I consider this is a well-presented paper with a significant impact on the specific field. Now, I believe this MS is now suitable for publication in PlosNTD.

Reviewer \#3: The authors have clearly improved the quality of the manuscript. They have qualified their comments about the EA patient. All my previous concerns were solved. The authors provided us a clear and innovative manuscript. Thank you.

\*\*\*\*\*\*\*\*\*\*

PLOS authors have the option to publish the peer review history of their article ([what does this mean?](https://journals.plos.org/plosntds/s/editorial-and-peer-review-process#loc-peer-review-history)). If published, this will include your full peer review and any attached files.

If you choose "no", your identity will remain anonymous but your review may still be made public.

**Do you want your identity to be public for this peer review?** For information about this choice, including consent withdrawal, please see our [Privacy Policy](https://www.plos.org/privacy-policy).

Reviewer \#1: Yes: Klaus Brehm

Reviewer \#2: No

Reviewer \#3: No

10.1371/journal.pntd.0008148.r004

Acceptance letter

Casulli

Adriano

Deputy Editor

Koziol

Uriel

Guest Editor

© 2020 Casulli, Koziol

2020

Casulli, Koziol

This is an open access article distributed under the terms of the

Creative Commons Attribution License

, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

2 Apr 2020

Dear Mr Ji,

We are delighted to inform you that your manuscript, \"Comprehensive characterization of plasma cell-free *Echinococcus* spp. DNA in echinococcosis patients using ultra-high-throughput sequencing.,\" has been formally accepted for publication in PLOS Neglected Tropical Diseases.

We have now passed your article onto the PLOS Production Department who will complete the rest of the publication process. All authors will receive a confirmation email upon publication.

The corresponding author will soon be receiving a typeset proof for review, to ensure errors have not been introduced during production. Please review the PDF proof of your manuscript carefully, as this is the last chance to correct any scientific or type-setting errors. Please note that major changes, or those which affect the scientific understanding of the work, will likely cause delays to the publication date of your manuscript. Note: Proofs for Front Matter articles (Editorial, Viewpoint, Symposium, Review, etc\...) are generated on a different schedule and may not be made available as quickly.

Soon after your final files are uploaded, the early version of your manuscript will be published online unless you opted out of this process. The date of the early version will be your article\'s publication date. The final article will be published to the same URL, and all versions of the paper will be accessible to readers.

Thank you again for supporting open-access publishing; we are looking forward to publishing your work in PLOS Neglected Tropical Diseases.

Best regards,

Serap Aksoy

Editor-in-Chief

PLOS Neglected Tropical Diseases

Shaden Kamhawi

Editor-in-Chief

PLOS Neglected Tropical Diseases

[^1]: The authors have declared that no competing interests exist.


Q:

Multiplying Matrices by Using Pointers in C

I am trying to create a method which is like this:
void Multiply(double *A, double *B, double *C, int N);
It is a function that computes C = A * B.
A, B and C are NxN(square) matrices and I have to do it by pointers.
The code I have written is like the following:
int Multiply(double *A, double *B, double *C, int N){
    int i,j,k;
    double sum;
    for (i = 0; i < N; i++) {
      for (j = 0; j < N; j++) {
         sum = 0;
         for (k = 0; k < N; k++) {

            sum = sum + (*(A+i*N+k)) * (*(B+k*N+i));
         }
         *(C+i*N+j) = sum;
      }
   }
}

It doesn't cause any errors and also it is not working.
I can't see the problem. Please can anybody help?

A:

i,j element of C is the row i of A multiply by the j column of B. The kernel of your loops should be:
sum = sum + (*(A+i*N+k)) * (*(B+k*N+j));


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Q:

How Do I group entries by Category and retain fixed Category Order?

I found a solution to grouping entries by category here which is working perfectly but I need the categories in the order they were arranged in the categories tab in the Admin panel.
Currently, the categories are outputting in the order they were entered but they've been re-ordered since then.
I tried using .fixedOrder(true) in a few places but nothing good happened. This is the current code:
{% set entries = craft.entries({
  section: 'projects'
}).find() %}

{% for category, catEntries in entries|group('projectCategory.first().title') %}

{{ category }}

        {% for entry in catEntries %}

{{ entry.title }}

        {% endfor %} {# -- // for entry -- #}

{% endfor %}{# -- // for category -- #}

A:

Depending on your needs, you may be able to ditch the group() filter and sort your list however you need.
Get all your entries and categories. Then use the relatedTo functions to filter those lists.
{% set categories = craft.categories.group('projectCategories') %}
{% set projects = craft.entries.section('projects').order('title').limit(null) %}

{% for category in categories.relatedTo(projects) %}

{{ category.title }}

    {% for entry in projects.relatedTo(category) %}

{{ entry.title }}

    {% endfor %}

{% endfor %}

One caveat might be that this solution pulls all your entries and categories. This worked for my situation where I had around 50 categories and under 200 entries. I also knew these numbers wouldn't get bigger in the future.
This also makes a few other assumptions too...

You DON'T need to show entries with NO related categories like this:

Category A

entry
entry

Category B

entry

(No Categories)

entry

Also, your entries can show up under EACH category they're related to, instead of just the FIRST category like your original code. For example, if Entry 1 has Category A and Category B selected, you'll get this:
Category A

entry 1

Category B

entry 1

A:

Twig is a templating language, and in my opinion this is pushing what it's comfortably meant to do. You might want to consider handling the heavy lifting w/ PHP by writing a small business logic plugin, or take a look at third party plugins like Micheal Rog's SuperSort to help out.
That being said, if you want to go plugin-less the following should work (note that you'll need to change "myProjectCategoryHandle" to the actual handle of the category group used for the projectCategory field):
{# Pull entries, group by category field "projectCategory" #}
{% set entries = craft.entries.section( 'projects' ) | group( 'projectCategory.first().title' ) %}

{# Get all category titles for the category group "yourProjectCategoryHandle" #}
{% set categories = craft.categories.group( 'yourProjectCategoryHandle' ).titles %}

{# Loop through the category title array #}
{% for category in categories %}

    {# Get the entries grouped under the current category title (if any) #}
    {% set catEntries = entries[ '' ~ category ] | default( [] ) %}

    {# Print the category title and entries #}
    {% if catEntries | length %}

{{ category }}

        {% for entry in catEntries %}

{{ entry.title }}

        {% endfor %} {# -- // for entry -- #}

    {% endif %}

{% endfor %}


Q:

Mean value theorem understanding

So I have this question here which says:
Say that f is differentiable and $f'(x)\neq1$ on $(-\infty,\infty)$. Show that there is at most one real number $a$ such that $f(a)=a$
I'm supposed to use the mean value theorem with the function $g(x)=f(x)-x$ but i'm not really sure how I'm supposed to incorporated the mean value theorem into this...
I tried to substitute for $f(b)$ with $g(b)+b$ and simplify things but i'm not getting anywhere.
Any help on this please?

A:

Suppose to the contrary there are two distinct values such that $f(a)=a$, and $f(b)=b$.
The defining $g(x)=f(x)-x$, we get $g(a)=0$ and $g(b)=0$.
By Mean Value Theorem (Note $g$ is also differentiable):
$g'(c)=\frac{g(b)-g(a)}{b-a}=0$ for some $c$ between $a$ and $b$.
This means $0=g'(c)=f'(c)-1$, so $f'(c)=1$, a contradiction.

A:

It is not necessary to consider $g(x)=f(x)-x$.
Suppose that there are $a,b \in \mathbb R$ such that $b>a$,  $f(a)=a$ and $f(b)=b$. Then
$\frac{f(b)-f(a)}{b-a}=1 \ne f'(c)$ for all $c \in (a,b)$, a contadiction


Higher visual functions, including object recognition and global motion detection, are carried out by extrastriate visual areas, or higher visual areas (HVAs), downstream from primary visual cortex. These higher processing functions often fail to completely recover following congenital blindness or amblyopia. These observations suggest that early visual experience sculpts HVA circuitry, but this has not been determined. To address this gap in knowledge, we propose an integrative approach using genetically engineered mice. Our goal is to determine how experience sculpts the normal development of HVA circuitry and how circuit deficits are caused by visual deprivation. Our preliminary data indicates that a subset of HVAs is slow to develop after eye opening, and fails to completely recover visual responses following dark rearing. By contrast, a complementary set of HVAs is visually responsive at eye opening, and fully recovers following dark rearing. We will map the development of HVAs at multiple time points, starting at eye opening, using intrinsic signal optical imaging and two photon calcium imaging. We will use a paradigm we developed for high resolution receptive field mapping of local populations of neurons in parallel. We will use technology we have recently developed to examine activity correlations between visual cortical areas and map how these change in development. We will dark rear mice to determine the effect of visual deprivation on HVA circuitry. The results from this project will reveal the role of visual experience in sculpting HVA selectivity and circuit development.