different sequence lengths detected, this will interfere with Stacks algorithms, trim reads to uniform length (override this check with --force-diff-len)

[E Ra]

Hi everyone,

I am trying to use denovo_map.pl (stacks v 2.61) to perform de novo analysis as described at here. My data consists of single-end samples with trimmed reads higher than 50 bases of length. denovo_map.pl seems to have an issue to deal with reads with different length and suggest using the option --force-diff-len.
Can someone tell me where to use this option or how to resolve this issue?
Thanks,

--Elie

These are my command lines:
samples="/home/00_test/02_trimmed/"
popmap=${samples}popmap_test.txt
output="/home/00_test/03_denovo_M2/"
denovo_map.pl --samples $samples --popmap $popmap --out-path $output --min-samples-per-pop 0.8 --rm-pcr-duplicates -M 2 -n 2

Here is the content of the log file:
ustacks parameters selected:
  Input file: '/home/00_test/02_trimmed/F1_24.fq.gz'
  Sample ID: 1
  Min depth of coverage to create a stack (m): 3
  Repeat removal algorithm: enabled
  Max distance allowed between stacks (M): 2
  Max distance allowed to align secondary reads: 4
  Max number of stacks allowed per de novo locus: 3
  Deleveraging algorithm: disabled
  Gapped assembly: enabled
  Minimum alignment length: 0.8
  Model type: SNP
  Alpha significance level for model: 0.05

Loading RAD-Tags...1M...
Error: different sequence lengths detected, this will interfere with Stacks algorithms, trim reads to uniform length (override this check with --force-diff-len).

denovo_map.pl: Aborted because the last command failed (1).
Last command executed by denovo_map.pl was:
  /usr/local/bin/ustacks -t gzfastq -f /home/00_test/02_trimmed/F1_24.fq.gz -o /home/00_test/03_denovo_M2 -i 1 -M 2

[Angel Rivera-Colón]

Greetings,

The --force-dif-len flag is specific to ustacks, therefore when using it with denovo_map you have to specify it using the -X flag (used to call specific options from the pipeline components). For example:

    denovo_map.pl [other options] -X "ustacks: --force-diff-len"

-Angel