README.md

AA_Metabarcoding

Using my own R package MultiAmplicon to analyse Multi-Marker Amplicon sequencing (aka Metabarcoding) data generated on the Fluidigm AccessArray.

Taxonomic annotation

Taxonomic annotation is a core component of downstream analyses and is achieved based on RDP classifiers trained on an expanded Silva database.

For Version 0.1 (Not for the current pipeline!)

An overview of a metabarcoding pipeline for multiple marker data from the Fluidigm Access Array. The present version is intended for a overview of the preliminary processing of data. Code can be reviewed in the /scripts and /R folders. The processing is not reproducible at the present version as raw data files cannot be accessed (yet).

Preprocessing

stratify the data into amplicons and samples

all samples in one file with sample identifier:

scripts/combine_and_lable_samples.pl *R1.fastq.gz > ALL_R1.fastq
scripts/combine_and_lable_samples.pl *R2.fastq.gz > ALL_R2.fastq

sort into amplicons

scripts/sort_amplicons.pl

run the usearch pipeline

parallel scripts/pipeLine.sh {} ::: /path/to/sorted/*.fastq

a little fix befor import of otu tables table for R

sed -i 's/#OTU ID/OTUID/ig' *.fastq.otu_table.txt

concatenate all otus

gawk '{if(/^>/){print $0"|"FILENAME} else{print $0}}' *.fastq.otus.fa > ALL_outs.fa

blast all otus against a silva subset of nt

blastn -query ALL_outs.fa -evalue 1e-20 -perc_identity 97 -db /SAN/db/blastdb/nt/nt -gilist /SAN/db/blastdb/silvaEukarya/SilvaEuk.gi -outfmt 11 > ALL_outs_vs_SilvaEuk.asn1

get the taxonomy based on these blasts

scripts/blast2alltax_outfmt11.pl ALL_outs_vs_SilvaEuk.asn1 > ALL_outs.taxtable

or instead based on a Megan lca analysis

/tools/MEGAN6/tools/blast2lca -i ALL_outs_vs_NT.xml -m BlastN -tn false -a2t /SAN/db/MEGAN/nucl-acc2taxid-August2016.abin -mid 97 -o ALL_outs_vs_NT.megantax