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Riaz.md

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An example on using other eDNA primers

Here we use the primer Riaz as an example.

Amplicon sequencing data

Amplicon sequencing data were from Brys et al, 2021. We choose pooled samples in timepoint T2 (SRR11479674~SRR11479774, 15 runs) as an example. These 15 accession IDs are:

SRR11479674
SRR11479675
SRR11479676
SRR11479677
SRR11479678
SRR11479680
SRR11479681
SRR11479682
SRR11479683
SRR11479769
SRR11479770
SRR11479771
SRR11479772
SRR11479773
SRR11479774

Download these data.

$ cd test
$ wget ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR114/074/SRR11479674/SRR11479674_1.fastq.gz
$ wget ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR114/074/SRR11479674/SRR11479674_2.fastq.gz
...
...
...
$ mkdir seq_riaz
$ mv SRR*.fastq.gz seq_riaz/

RefDB

In this example, we directly use the local RefDB provided by Brys et al, 2021. Download it and changed to the MiFish RefDB format. See test/INBO_riaz.db.fa as an example.

In other situations, if you do not have a local refDB for your primers, you can follow CRABS to make a refDB (step 1~6, using MitoFish as original source), then using the awk command to change it to FASTA format.

$ awk '{print ">gb|" $1 "|" $9 "\n" $10}' output.tsv >your.db.fa
$ makeblastdb -dbtype nucl -in your.db.fa

Run Pipeline

The primer sequence of Riaz is

FWD = 5′-ACTGGGATTAGATACCCC-3′
REV = 5′-TAGAACAGGCTCCTCTAG-3′

Try running

$ mifish seq_riaz INBO_riaz.db.fa -m 0 -M 99999 -f ACTGGGATTAGATACCCC -r TAGAACAGGCTCCTCTAG -s -t 10

It should generate results the same as test/MiFishResult-example-Riaz. Species composition are in consistance with the result in Figure 2, Brys et al, 2021