Quality score after Illumina run - should it be coverted before samtools and gatk?

[gfmgfm]

Hi all,

I am a bit confused with the base quality score. After Illumina run, I use BWA to map the reads. I then use downstream applications such as samtools and gatk. But samtools accepts sanger phred (ASCII-33), while Illimina's fastq is ASCII-64. Is it correct that the fastq need to be converted before running BWA to sanger scores? If so, what tools do people use for this?

[csoong]

yes, you should convert first, even before BWA.
google for fq_all2std.pl

[gfmgfm]

thanks a lot!

[gfmgfm]

I took a look at the code of fq_all2std.pl . I believe I need the command:
sol2std Convert Solexa/Illumina FASTQ to the standard FASTQ

But doesn't it convert the old Illumina (prior to version 1.3) to sanger?

The conversion table it uses:
for (-64..64) {
$conv_table[$_+64] = chr(int(33 + 10*log(1+10**($_/10.0))/log(10)+.499));
}

[Seq84]

But doesn't it convert the old Illumina (prior to version 1.3) to sanger?

Yeah this script converts only solexa format and not illumina(1.3 or 1.5+) one.

[trickytank]

BWA option -I

In the recent versions in BWA you no longer need to convert the quality scores. From the BWA manual:

-I The input is in the Illumina 1.3+ read format (quality equals ASCII-64).

[mboursnell]

So it seems you don't need to convert before BWA. Do you need to convert before GATK or before samtools?

[trickytank]

There isn't any need to, as all SAM/BAM files have QUAL field: "ASCII of Phred-scaled base QUALity+33". This way you don't need to worry about the quality scale in SAM/BAM.

(Note that it is possible to get qualities in a SAM/BAM like file that are scaled QUALity+64, BUT these are not real SAM/BAM files. BWA produces a proper SAM/BAM file with the correct quality scale.)

[MissDona]

what is the consequence of losing -I option?

As asked in the title, what if I missed the -I option while run bwa? is there any remedial work I can do to save it? thx!

[gfmgfm]

Current Illumina's qualities in the fastq files changed since the first post on this issue. They are now (Illumina 1.8+) Phred+33, and therefore no need to specify -I for BWA. Just make sure the fastq files you are working with are indeed Illumina 1.8+.