bat guano , an excrement of the cave - dwelling bats forms the basis of the ecology inside the cave by acting as a food source for detritivorous microbes .
it contains high content of organic carbon , nitrogen , phosphate , and potassium , .
bacteria present in bat guano were reported to be involved in nitrification process and were also known as potential chitinase producer .
a clone library based study in bat guano samples has revealed the presence of group 1.1a and 1.1b crenarchaeota , an efficient ammonia oxidizer , .
analyzing bat guano is also important since they often harbor various pathogens which can be thread for speleologists , and tourists .
although the microbial communities in diverse cave ecosystems have been studied , little is known about the microbial communities of bat guano heaps ,   and there has been no studies using high throughput sequencing technology .
meghalaya is known to possess the largest and most diverse karst caves in the world .
pnahkyndeng cave located in ri - bhoi district of meghalaya , india is a home of various bats and offering an ideal environment for studying the bat guano microbiota without any anthropological influence .
samples were collected on february 2014 from the bat guano of pnahkyndeng cave ( 255722.70n , 915543.10e ) , nongpoh , ri - bhoi district , india .
ten composite guano samples were collected from different places of the cave floor and the soil community dna was extracted separately using the fast dna spin kit for soils ( mp biomedical , solon , oh , usa ) . the freshly extracted dna was purified twice using 0.5% low melting point agarose gel and mixed to prepare a composite sample .
final dna concentrations were quantified by the using a microplate reader ( bmg labtech , jena , germany ) .
the v4 region of the 16s rrna gene was amplified using f515/r806 primer combination ( 5-gtgccagcmgccgcggtaa-3 ; 5-ggactachvgggtwtctaat-3 ) .
amplicon was extracted from 2% agarose gels and purified using the qia quick gel extraction kit ( qiagen , valencia , ca , usa ) according to the manufacturer 's instructions .
quality filtering on raw sequences was performed according to base quality score distributions , average base content per read and gc distribution in the reads .
singletons , the unique otu that did not cluster with other sequences , were removed as it might be a result of sequencing errors and can result in spurious otus .
chimeras were also removed using uchime and pre - processed consensus v4 sequences were grouped into operational taxonomic units ( otus ) using the clustering program uclust at a similarity threshold of 0.97 , .
all the pre - processed reads were used to identify the otus using qiime program for constructing a representative sequence for each otus .
the representative sequence was finally aligned to the greengenes core set reference databases using pynast program , .
, 403,529 reads were classified at the phylum , 282,350 at the order , 188,406 at the family and 2926 sequences were classified at the species levels .
classified otus belonged to 18 different phyla dominated by chloroflexi , crenarchaeota , actinobacteria , bacteroidetes , proteobacteria , and planctomycetes ( fig .  1 ) .
analysis of bacterial communities revealed the two most dominant bacteria 's  chloroflexi ( 29.97% ) and actinobacteria ( 22.55% ) , which are known to be a common inhabitant of cave microflora .
other identified phyla include crenarchaeota ( 16.96% ) , planctomycetes ( 12.41% ) and proteobacteria ( 12.03% ) .
chloroflexi was divided into 11 classes  thermomicrobia , planctomycetia , gitt - gs-136 , ktedonobacteria , anaerolineae , tk10 , tk17 , s085 , chloroflexi , ellin6529 , and gitt - gs-136 .
the most dominant otu within this phyla was denovo 317 , classified under the class thermomicrobia ( 40.52% ) followed by denovo 710 under the thermomicrobia ( 7.61% ) , denovo 235 under the genus thermogemmatisporaceae ( 7.18% ) and denovo 3 under the genus gemmataceae ( 2.98% ) .
the dominant otu within this phyla was denovo 74 under the genus mycobacterium ( 29.39% ) followed by denovo 993 ( 8.27% ) and denovo 372 ( 5.27% ) classified under the genus acidimicrobiales and actinomycetales , respectively .
only five otus were classified up to the species level ( mycobacterium llatzerense and mycobacterium celatum ) .
a third dominant phylum in this sample was identified as planctomycetes comprising of 91 otus and 46,063 reads .
seventeen archeal otus were classified under the order nrp - j , methanomicrobiales and methanosarcinales .
four of them were identified at the genus level ( methanosarcina , haloquadratum , methanosaeta and methanocorpusculum ) .
the phylogenetic tree based on the genus level relationships is provided as supplementary fig .  1 .
previous study on archaeal communities present in bat guano identified many ammonia oxidizing bacteria but it was limited with a few number of clones .
our analysis provides in - depth and high throughput identification of the bacterial communities present in bat guano . in the present study
, we identified 18 bacterial phyla and most of the bacterial genus identified was known to be involved in nitrogen cycling as seen in previous study .
a significant portion of otus still remains unclassified which indicates the possibility for the presence of novel species in pnahkyndeng cave .
further studies like whole metagenome sequencing or functional metagenomics can illustrate the detailed information of this bacterial community .
 v4 hypervariable region of 16s rdna was analyzed for identifying the bacterial communities present in bat guano from the unexplored cave  pnahkyndeng , meghalaya , northeast india . 
 metagenome comprised of 585,434 raw illumina sequences with a 59.59% g+c content . 
 a total of 416,490 preprocessed reads were clustered into 1282 otus ( operational taxonomical units ) comprising of 18 bacterial phyla . 
 the taxonomic profile showed that the guano bacterial community is dominated by chloroflexi , actinobacteria and crenarchaeota which account for 70.73% of all sequence reads and 43.83% of all otus . 
 metagenome sequence data are available at ncbi under the accession no . 
 srp051094 . 
 this study is the first to characterize bat guano bacterial community using next - generation sequencing approach . 
Direct link to deposited data
Experimental design, materials and methods
Nucleotide sequence accession number
Competing interests