Fastq Qualities For Solexa/Illumina

Question

[Pierre Lindenbaum]

The FASTQ Format Specification says:

Although Solexa/Illumina read file looks pretty much like FASTQ, they are different in that the qualities are scaled differently. In the quality string, if you can see a character with its ASCII code higher than 90, probably your file is in the Solexa/Illumina format.

Should I care about this when I use some tools such as MAQ, SAMTOOLS, GATK, etc... ?

Answer 1

[Brad Chapman]

Absolutely. There are, annoyingly, 3 different fastq quality encodings. The wikipedia page is a very good resource: http://en.wikipedia.org/wiki/FASTQ_format and this paper from several OpenBio folks: http://nar.oxfordjournals.org/cgi/content/abstract/38/6/1767

All aligners that use quality scores will have a flag to specify which encoding your fastq file is in. The approach Galaxy takes is to use a groomer to convert everything to Sanger format, and then work from there: http://main.g2.bx.psu.edu/u/dan/p/fastq

[Louis Letourneau]

Actually BWA doesn't really, but samtools expects phred+33 quals, if that's not what it gets, the pileup caller and varfilter will not give good results (they'll be over confident)

Answer 2

[Istvan Albert]

Usually you will need to pass a flag to specify the encoding type. When using the bowtie aligner for example you will have to pass the flag:

--phred64-quals

For some tools you may need to convert them yourself.

PS. The Illumina software with version prior to 1.3 uses a different formula for producing the quality measures

Answer 3

[Casey Bergman]

The FASTX toolkit has an undocumented option to set the offset for the ascii quality conversion. The default offset is 64 (i.e. Illumina quality encoding) but this can be overridden with the -Q option, e.g.:

$ fastq_to_fasta -n -Q 33 -i in.fq -o out.fa

See: http://seqanswers.com/forums/showthread.php?t=6701