Two peaks on FastQC plot "Per sequence GC content"

[nucacidhunter]

Would you be able to post all of the FastQC output plots for comparison with other runs. For now, I would mention that Exome capture does not sample genome randomly, so it is not unusual to see what you are reporting.

[Khillo81]

Thanks for your response. I first have to mention that I don't have a very strong background in bioinformatics and am using the CLC Genomics Workbench (ver. 7.5) which has a GUI and runs on Windows. I have used the Workbench's 'Merge Overlapping Pairs' function to generate the histogram below (I'm guessing it's similar to the BBMerge mentioned by Brian). I also haven't used the FASTQC but the native QC check in the Workbench. I'm attaching the output here. As you can see there is no severe drop in quality along the reads and besides the peaks in GC content observed at the end of the read (as I understand it, typical for Illumina data), the GC content along read length is around 45%. And the samples are human.

[Brian Bushnell]

Unfortunately, it looks like that tool does not merge reads with insert size shorter than read length, which was the point of the exercise. But from the graph I can infer that maybe 30% of the reads are indeed in that category, so there are a few possibilities:

1) The twin peaks are indeed from exon-capture bias, though I kind of doubt that, as it does not explain why trimming the reads would reduce it; and I would have expected such a bias to shift the peak center rather than creating a bimodal distribution, but of course it depends on the bait design.
2) There is an exonic and intronic peak, or gene and non-gene peak. The GC content of a gene changes markedly once you get just outside of its bounds. For example, just upstream of the gene, it becomes very AT-rich, IIRC. But, I don't really like that explanation either.
3) The adapter-trimming is unsuccessful or incomplete. From your GC content by base position, it looks fairly flat across the read, aside from the first 20 bp... so that doesn't make much sense either. Still, it wouldn't hurt to confirm. What were the total percent of reads and bases trimmed during adapter-trimming? I would expect something like 30% of the reads and maybe 5-10% of the bases. If you are using Nextera adapters, be sure you use those sequences for trimming.


I suggest that you bin some of your reads by GC - just split them into pairs with GC<50% and GC>50%. Map both to human and look at the mapping rates (ideally, forcing unclipped global alignments). If they are equivalent, then the issue is not caused by contamination or adapter sequence, and it's probably safe to ignore.

You can split the reads by GC content with my reformat tool:

reformat.sh in1=read1.fq in2=read2.fq out1=low1.fq out2=low2.fq maxgc=0.5

reformat.sh in1=read1.fq in2=read2.fq out1=high1.fq out2=high2.fq mingc=0.5

[Dr khani]

my fastq GC content report has two peaks.can any one help me how i can assemble these type of data?

[luc]

As mentioned above, the two peaks could very well be a sign of a mixed sample (contamination).
You could remove the all the high GC content reads and see if this improves the assembly.
BBtools (BBduk?) has a GC content filter.

[Dr khani]

thank you . i can not run bbmap tools on windows. i get error