marinemoleco / spongeemp_blastdb

BLAST database - sponge EMP deblurred subOTUs 1.0

How to cite

This sponge EMP blast database has been created for a recent publication. If you use the database, please cite this article:

Dat TTH, Steinert G, Thi Kim Cuc N, Smidt H, Sipkema D. (2018) Archaeal and bacterial diversity and community composition from 18 phylogenetically divergent sponge species in Vietnam. PeerJ 6:e4970 https://doi.org/10.7717/peerj.4970

spongeEMP_BLASTdb 1.0

SpongeEMP deblurred subOTUs BLAST database

1.) Sequence data source

The sponge microbiome project (see https://academic.oup.com/gigascience/article/6/10/1/4082886) deblurred biom file "final.withtax.biom" was downloaded from https://github.com/amnona/SpongeEMP/tree/master/sponge_emp/data

2.) Biom to tab separeted text file transformation

The biom file was converted to a tsv file:

biom convert -i final.withtax.biom -o final.withtax.tsv --to-tsv --header-key taxonomy

Subsequently, the stored OTU representative sequence information was extracted from this text file and stored in a separated fasta file "final.withtax.v12.fa"

3.) Representative sequence classification using Silva128

Representative sequences for each deblurred OTU was classified using mothur v.1.39.5 https://www.mothur.org/wiki/Download_mothur and silva128 https://www.mothur.org/wiki/Silva_reference_files#Release_128

a) First just a simple classification without chimera search and removal of Mitochondria-Chloroplast-Eukaryota-unknown sequences

classify.seqs(fasta=final.withtax.v12.fa, template=silva.nr_v128.align, taxonomy=silva.nr_v128.tax, cutoff=80, probs=T, processors=6)

taxlevel rankID taxon daughterlevels total 0 0 Root 4 83908 1 0.1 Archaea 10 1025 1 0.2 Bacteria 53 64429 1 0.3 Eukaryota 13 915 1 0.4 unknown 1 17539

b) Chimera search Creating name file to use EMP sequences as template for chimera search

unique.seqs(fasta=final.withtax.v12.fa)

Chimera search and removal

chimera.vsearch(fasta=final.withtax.v12.fa, name=final.withtax.v12.names, dereplicate=t, processors=6)

Using 6 processors. Checking sequences from final.withtax.v12.fa ... When using template=self, mothur can only use 1 processor, continuing. vsearch v2.3.4_linux_x86_64, 15.6GB RAM, 8 cores https://github.com/torognes/vsearch

Reading file final.withtax.v12.temp 100%
8390800 nt in 83908 seqs, min 100, max 100, avg 100 Masking 100%
Sorting by abundance 100% Counting unique k-mers 100%
Detecting chimeras 100%
Found 0 (0.0%) chimeras, 83908 (100.0%) non-chimeras, and 0 (0.0%) borderline sequences in 83908 unique sequences. Taking abundance information into account, this corresponds to 0 (0.0%) chimeras, 83908 (100.0%) non-chimeras, and 0 (0.0%) borderline sequences in 83908 total sequences.

It took 27 secs to check 83908 sequences. 0 chimeras were found.

c) emoval of certain sequences Removing Mitochondria-Chloroplast-Eukaryota-unknown sequences from seq file

remove.lineage(fasta=final.withtax.v12.fa, taxonomy=final.withtax.v12.nr_v128.wang.taxonomy, taxon=Mitochondria-Chloroplast-Eukaryota-unknown)

Output File Names: final.withtax.v12.nr_v128.wang.pick.taxonomy final.withtax.v12.pick.fa

final.withtax.v12.fa 83908 OTUs final.withtax.v12.pick.fa 64424 OTUS

4.) Creating the local spongeemp BLAST database

a) Go to the BLAST download folder and download the BLAST executables for your operating system: ftp://ftp.ncbi.nlm.nih.gov/blast/executables/blast+/LATEST/ - unzip this archive.

b) Copy the fasta file into the ncbi-blast-x.x.x bin folder.

c) Open your terminal and go to the bin folder. Use the following command to create the spongeemp nucleotide BLAST database:

makeblastdb -in final.withtax.v12.pick.fa -out DeblurV12PickOTUsSilva128 -dbtype 'nucl' -hash_index