the rapid improvement in quality , quantity , and cost of next generation sequencing ( ngs ) has resulted in commensurate improvements in analysis techniques . for bacteria ,
the availability of kits for library preparation , rapid and high content sequencing , and mature data analysis pipelines for genome resequencing and assembly had drastically reduced costs and improved reliability of these results .
the commoditization of bacterial genome sequencing has led to more complex applications : clinical and agricultural diagnostics [ 14 ] , outbreak detection and monitoring [ 57 ] , human health studies [ 8 , 9 ] , biocatalysis [ 10 , 11 ] , environmental studies , and many others [ 13 , 14 ] . for ngs platforms ,
current sequencing technologies require that sequencing adapters be ligated to dna fragments before sequencing is possible .
ligation of adapters to ( typically small ) dna fragments is an inefficient process , generating ligated hybrids from only a small fraction of targeted dna molecules .
this limitation in turn increases the required dna input , with the only goal being to generate sufficient numbers of ligated fragments to allow sequencing .
typical library preparation methods require large amounts ( ~1  g ) at high concentrations ( > 25  ng / ml ) of dna for successful library generation , limiting the types of samples that can be sequenced reliably .
these include high variability of evenness and completeness of genome coverage as a function of % gc content , input dna quantities , and sequencing technology [ 1519 ] . these impact the amount of sequencing data required and the quality of genome assembly and analysis .
several library preparation kits that require 1100  ng of input dna are now available ( new england biolabs ' nebnext , illumina 's truseq nano , bioo scientific 's nextflex , nugen 's ovation ultralow , etc . ) .
this paper details the results of evaluation of the utility of the nebnext ultra library preparation kits for both resequencing and assembly of several bacterial genomes .
we compare the evenness and completeness of coverage between nebnext ultra and illumina truseq kits for bacterial genomes of varying size and % gc content .
our findings indicate that low dna input amounts are sufficient to generate high quality sequencing data that can be used for genome resequencing or de novo assembly ( if combined with long fragment data ) .
we sequenced three different bacterial species with various genome lengths ( from 5.4  mb to 6.7  mb ) and containing various % gc contents ( from 35% to 68% ) .
standard input dna amounts were 100  ng , approximately 10x lower than the required amount for the illumina truseq kit and 10x higher than the minimum dna inputs per nebnext ultra manual specifications .
the most challenging ( longest genome and highest gc content ) bacterial genomes ( burkholderia a and b ) were also sequenced with minimal dna inputs ( 10  ng ) .
all samples were sequenced on the illumina hiseq platform using 2  100  bp chemistry .
data analyses consisted of read - mapping the short fragment data to reference genomes using bwa ( burrows - wheeler alignment ) .
these data were also combined with long insert mate pair data to evaluate their utility for de novo assembly of the bacterial genomes .
genomic dna from bacillus anthracis ( strain sterne 34f2 ) was isolated from a log phase culture using the mo - bio ultraclean microbial dna isolation kit .
burkholderia thailandensis a ( strain e254 , accession numbers cp004381 and cp004382 ) and burkholderia thailandensis b ( strain usamru malaysia # 20 , accession numbers cp004383 and cp004384 ) are previously reported strains , and dna was provided by dr .
the integrity of all genomic dna samples was evaluated using agarose gels and their quantity measured with picogreen reagents on a qubit 2.0 instrument .
nebnext ultra library preparation protocol consists of several enzymatic and two purification steps , one of which is used for size selection of library fragments .
genomic dna samples were sheared in 55  l of tle buffer ( 10  mm tris , 0.1  mm edta , ph 8) using covaris e220 with the following settings : duty cycle 10% , intensity 5 , cycle 200 , and time 100  sec .
after shearing , two enzymatic steps ( end preparation and adapter ligation ) are performed in the same tube , followed by size selection of the library fragments using a double ampure cleanup .
first ampure step used 0.4x sample volume of beads and the supernatant was transferred to a clean tube .
selected library fragments were amplified with barcoded primers ( 1012 pcr cycles ) and purified one more time with ampure beads ( 0.5x bead volume ) ( see supplementary material available online at http://dx.doi.org/10.1155/2014/434575 ) .
nebnext libraries were analyzed using bioanalyzer 2100 and dna 1000 or dna high sensitivity chips , to quantify the library size and assess the level of adapter - dimer and primer - dimer contamination .
libraries were quantified using illumina library qpcr quantification kits from kapa biosystems and sequenced on either the illumina miseq or illumina hiseq .
the illumina data from this study were trimmed to remove any ambiguous bases ; any reads shorter than 70  bp after trimming and the corresponding read pairs were discarded .
the total number of reads per sample ranged from 6.2 million to 47.8 million before trimming .
all data had read lengths of 151  bp with one exception which had read lengths of 101  bp . after trimming , the average read lengths were reduced by less than 3.5% for all samples .
the data for each sample were normalized to 70x coverage of the genome after trimming .
the average number of reads with a quality greater than q20 after trimming and normalization ranged from 61% of the total reads to 97% of the total reads .
the total number of reads , the number of reads with quality greater than q20 , and the average read lengths before and after trimming for each sample can be found in table s1 .
the assemblies were compared to the reference genomes to consider insertion / deletion errors and rearrangements using an in - house perl script . for read - mapping ,
burrows - wheeler alignment ( bwa ) mapping tool was used , combined with samtools and in - house perl scripts for coverage and insert size analysis [ 21 , 22 ] . for base coverage we used bwa global alignment option with default parameters .
bwa global alignment only reports the best alignment based on score calculated by a set of parameters .
if a read has several possible best alignment spots , bwa randomly assigns the read to one spot .
all reads mapped to contigs were used to calculate base coverage . for insert size calculation , only properly paired reads ( read pair on the same contig and with correct orientation ) were used .
we report the mean , standard deviation , the minimum , and maximum of the insert size distribution for all short fragment libraries .
we utilized three thresholds for reporting coverage : 0% , 1% , and 10% of mean fold coverage .
calculation of evenness of coverage was performed by calculating the average and standard deviation of coverage across nonoverlapping 10  kbp fragments of the finished genome .
evenness for each fragment was calculated as 1  ( standard deviation of coverage / coverage ) .
all data points ( genomic and plasmid coverage , where appropriate ) were used to generate box and whisker plots in ibm 's statistics program spss .
two debruijn graph assembly tools were used to evaluate the quality of the short fragment data for the purpose of assembling high quality genomes .
paired reads were randomly selected ( in silico ) from each sample to generate libraries of approximately 70-fold genome coverage for each sample , in order to normalize the data .
the only exception was the e. coli sample prepared with the truseq kit , for which only 61-fold coverage was available .
the 70-fold short fragment illumina data were combined with previously sequenced long insert mate pair data generated by 454 .
the 454 data had an average insert size of 8  kbps and provided 7- to 8-fold base coverage , with the exception again for the e. coli samples , which had approximately 3.5-fold coverage .
the library preparation protocol , as described in section 2 , yields average insert sizes of ~270  15  bps ( average library sizes of ~400  15  bps ) that are optimal for either 2  100 or 2  150  bp sequencing on illumina platforms .
different insert sizes can easily be obtained by adjusting the size selection step ( ratio of dna solution to ampure beads ) as recommended by the manufacturer .
it is not necessary to adjust the shearing step , as the sheared dna produced by covaris has a very broad size distribution .
nebnext library process provides very consistent results in terms of library size and concentration , even when performed for the very first time .
prior to normalization and sequencing , samples were analyzed using qubit ( picogreen - based method ) , bioanalyzer 2100 , and quantitative real - time pcr ( qpcr , kapa biosystems ) .
unfortunately , this was not the case when molar library concentrations were obtained with qubit and bioanalyzer data only ( without qpcr ) .
sequencing was performed on either illumina hiseq ( 2  100  bp ) or illumina miseq ( 2  150  bp ) . 

figure 1 ( b. anthracis and e. coli ) and figure 2 ( b. thailandensis a and b ) contain sliding window coverage plots that compare the coverage of each genome by different library preparation method and different dna input amount . from the figures , it can be seen that the genome coverage is remarkably similar regardless of the library preparation method ( nebnext or truseq ) .
of particular interest is that even the libraries prepared from only 10  ng of genomic dna produced essentially the same evenness of genome coverage as the rest of the samples ( figures 2(a)2(d ) , top panel ) .
as table 1 shows , the number of true gaps in coverage ( 0% ) is slightly higher for nebnext than for truseq libraries , while the number of gaps is lower for nebnext when using 1% or 10% average coverage thresholds .
the data were not normalized among all samples prior to evenness of coverage comparisons . instead , they were normalized within each sample relative to the average coverage . 

figure 3 shows box and whisker plots of the evenness of coverage of 10  kbp windows for each genome . in the case of e. coli ,
the evenness of coverage for nebnext ultra libraries prepared with 100  ng of input dna is superior to that of truseq libraries produced with 1  g of input dna . for b. anthracis and b. thailandensis ,
there is more variation in coverage for the nebnext preparations at both 100  ng and 10  ng . further examination of this effect suggests that it is proportional to the amount of input dna , supporting the theory that nebnext ultra kits either do not introduce bias or introduce similar bias to truseq kits , with a lower dna requirement . 

figure 4 shows the results of de novo genome assemblies generated using the short fragment data either alone ( figure 4(a ) , assembled with idba ) or complemented with long insert mate pair data ( figure 4(b ) , assembled with allpaths ) .
idba assemblies show very similar results for low ( b. anthracis ) to medium ( e. coli ) % gc genomes .
however , data obtained from nebnext libraries show a dramatic reduction in the number of contigs for high % gc genomes from the two burkholderia strains .
importantly , nebnext libraries prepared from just 10  ng of genomic dna maintain the high quality of genome assembly , producing similar numbers of contigs as with 100  ng input dna samples .
however , scaffolding mostly depends on the long insert mate pair data , which are the same for all assemblies .
comprehensive assembly statistics are shown in tables s2a and s2b . in conclusion , we have demonstrated that the quality of bacterial genome resequencing and de novo assembly is similar , regardless of the library preparation method and input dna amount ( from 10 to 1000  ng ) .
the only significant difference was observed in the assemblies of the b. thailandensis genomes , where nebnext library data produced dramatically more contiguous assemblies ( figure 4 ) . in general , the assemblies from the truseq libraries were more prone to indels and rearrangements .
the full results for the comparisons of our assemblies to the reference genomes are found in table 2 .
this is likely due to the improved ability of the nebnext reagents to more effectively amplify high % gc regions that are very common in burkholderia genomes .
modern library preparation methods for next generation sequencing technologies , as represented by nebnext ultra , are enabling bacterial genome sequencing from very small input amounts of genomic dna .
these methods likely do not require any additional improvement , since handling and quantifying dna amounts smaller than 10  ng can become challenging .
we have deposited the genomes of burkholderia thailandensis a ( strain e254 , accession numbers cp004381 and cp004382 ) and burkholderia thailandensis b ( strain usamru malaysia # 20 , accession numbers cp004383 and cp004384 ) into genbank .
all experimental and computational methods used during this work are publically available or can be provided by the authors .
 sequencing bacterial genomes has traditionally required large amounts of genomic dna ( ~1  g ) . 
 there have been few studies to determine the effects of the input dna amount or library preparation method on the quality of sequencing data . 
 several new commercially available library preparation methods enable shotgun sequencing from as little as 1  ng of input dna . in this study , we evaluated the nebnext ultra library preparation reagents for sequencing bacterial genomes . 
 we have evaluated the utility of nebnext ultra for resequencing and de novo assembly of four bacterial genomes and compared its performance with the truseq library preparation kit . 
 the nebnext ultra reagents enable high quality resequencing and de novo assembly of a variety of bacterial genomes when using 100  ng of input genomic dna . 
 for the two most challenging genomes ( burkholderia spp . ) , which have the highest gc content and are the longest , we also show that the quality of both resequencing and de novo assembly is not decreased when only 10  ng of input genomic dna is used . 
1. Introduction
2. Materials and Methods
3. Results and Discussion
4. Availability