filtering out low quality sequences from Sanger Sequencing

Question

[hassani.medamine]

Hello! I am currently facing a challenge of filtering out low quality sequences (output from Sanger sequecing, roughly I have 300 sequences, command line are very much appreciated).

Below, I explain what I have done so far, and I am looking for any hints/tips to get this working.

1- conversion ab1 to fastq " for filename in *.ab1; do seqret $filename fastq_files/$filename.fastq -osformat fastq; done"

2- trimming sequencing "for filename in *.fastq; do fastx_trimmer -f 30 -l 600 -i $filename -o t_$filename ; done"

3- trying to filter the sequences using " quality fastq_quality_filter -i input.fastq -o output/output.fastq -q 20 -v -Q 33 " and I got the following error message,

stack smashing detected : fastq_quality_filter terminated zsh: abort (core dumped) fastq_quality_filter -i PosKonR.fastq -o output/Pos.fastq -q 20 -v -Q 33

I changed to -Q 64 and got " fastq_quality_filter: Invalid quality score value (char '&' ord 38 quality value -26) on line 4 "

Many thanks in advance for any help on this

[h.mon]

Use Staden or Phred/Phrap. I think there are newer tools, but I am not up-to-date.

[GenoMax]

ClinQC which may be able to help. There is a also convertrix. It can automatically trim the untrusted regions (low quality bases) at the end of samples per its web page.

Answer 1

[hassani.medamine]

Thank you all, The issue was with output format from seqret. I have used instead fasta_to_fastq.py available here.