Many ### in fastq file (@HWI-ST...)?

[xhuister]

Hi all,

I got some sequencing files in fastq format, like this:

@HWI-ST741:189:C0GU5ACXX:8:1101:1219:1953 1:N:0:
GTGAGTTTTTTTTTTTTTTTTTTAAATTCAATAAAGAAAATTTAGTTAAAAACACAAATACAGATGAGTATGTCT
+
?@@?DDFFFFFIIIIIIIFD#####################################################
@HWI-ST741:189:C0GU5ACXX:8:1101:1156:1999 1:N:0:
TGGAGTTTTTTTTTTTTTTTTTTTTTAGACGGAAAGAGGGTTACAGAAGAAACACCGACACACATCGCATATGGC
+
@+=DDDDDHHHHHIIIIIIIHC#####################################################

This format is "Phred+33" fastq quality format, right? I checked this from WIKI (http://en.wikipedia.org/wiki/FASTQ_format).

What I confused is there are many reads (~50%) with many low quality scores ######... (score=35, which means a very low score 35-33=2).

Is this possible? Or is this the sequencing problem?

Thank you!

Wu

[swbarnes2]

You are interpreting that correctly. Those are reads whose quality drops to zilch after the first 20 or so bases. 2 is really as low as it goes. A 1 would technically means that you only have a 1 in 10 chance of the base being accurate, but obviously with only 4 nucleotides, the odds can't be that poor, hence, the quality is 2, represented by a # in phred +33

If most of the reads look like that, talk to whomever ran the instrument, because something bad happened during the run.

[xhuister]

You are interpreting that correctly. Those are reads whose quality drops to zilch after the first 20 or so bases. 2 is really as low as it goes. A 1 would technically means that you only have a 1 in 10 chance of the base being accurate, but obviously with only 4 nucleotides, the odds can't be that poor, hence, the quality is 2, represented by a # in phred +33

If most of the reads look like that, talk to whomever ran the instrument, because something bad happened during the run.

Thank you very much, swbarnes2. I've asked the guy who sequenced these samples, but he never replies. So I posted it here for help. Now I'm sure this is the problem, I'll check this with the sequencing people.

[maubp]

See this recent thread, http://seqanswers.com/forums/showthread.php?t=20709

The run of ##### at the end of a read's quality string (PHRED quality 2) means Illumina's software thinks there is a problem here. This is know as the "Read Segment Quality Control Indicator". Under the old Illumina FASTQ encoding Q2 was a 'B'.

[xhuister]

Thanks maubp. It is really the sequencing problem.