Fastq files quality

Question

[Lolla]

Hello, I have some fastq files, I found the base call quality for each sequence in the files then averaged them and plot the mean base call quality for each file. I noticed that for all files; there was drop in the middle of the sequences. My question is: is this an indication of artifact? have any of you experienced this? Any advice is appreciated..Thanks in advance..

[mbens]

Were all samples (files) sequenced on the same lane or in the same sequencing run? Generally, for read data obtained from Illumina sequencers, you would expect good quality in the midsection and lower quality at the ends of reads. So a quality drop in the midsection might indicate some failure (air bubbles, ...), but is not necessarily bad. You could go for further inspection of duplication levels or sequence composition (for instance using FastQC).

[shelkmike]

Please specify: 1) How deep is this drop (to what Q-score does the quality fall)? 2) Does the quality recover after this drop? I mean, does it remain low till the end of reads or it rises to normal values after the drop? 3) At what base from the beginning of reads does this drop occur? I mean, number, like 74th base

[Lolla]

Thanks shelmike; 1) it drops from 38+ to 34 or less in some files.. 2) yes, the quality increased after the drop point 3) 75th base

Answer 1

[shelkmike]

First of all, if it drops to 34 or so, this is not a problem - 34 is pretty good quality (it implies that the probability of error is about 0.0004) . I always see this drop in reads from HiSeq 2000. My colleagues explained it to me long ago - as far as I remember, this is because the sequencing machine refocuses on the flow cell during this cycle.

[Lolla]

That's true 34 still good but it was remarkable point as the drop happens in the same position almost in all samples..