Help Troubleshooting FASTQC
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3.0 years ago
mac03pat ▴ 30

My FASTQC raised three errors. Per base sequence content, per sequence gc content, and sequence duplication levels. I read that this could be because adapters have not been trimmed. I tried using Trimmomatic to trim adapters but I'm not sure that I did it correctly. I used Illuminaclip and tried both TruSeq3 options, I also did slidingwindow averaged bewtween 4 bases with a cutoff score of 20. The sequencing lab sent me a text file of the adapters and I tried cutting and pasting it in as a custom adapter but received an additional sequence length distribution error when I ran the FASTQC. Here's the text file contents: (I removed the > symbols because they were making the post weird but they preceded each "Adapter")

Adaptor1_5p GATCGGAAGAGCACACGTCTGAACTC

Adaptor2_5p AGATCGGAAGAGCGTCGTGTAGGGAA

Adaptor3_5p TTCCCTACACGACGCTCTTCCGATCT

Adaptor4_5p GAGTTCAGACGTGTGCTCTTCCGATC

Adaptor1_3p TTCCCTACACGACGCTCTTCCGATCT

Adaptor2_3p GAGTTCAGACGTGTGCTCTTCCGATC

Adaptor3_3p GATCGGAAGAGCACACGTCTGAACTC

Adaptor4_3p AGATCGGAAGAGCGTCGTGTAGGGAA

TruSeq-Universal_Adapter_5p AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT

TruSeq-Universal_Adapter_3p TCTAGCCTTCTCGCAGCACATCCCTTTCTCACATCTAGAGCCACCAGCGGCATAGTAA

Is this dual indexed?

Per base sequence content

Per sequence GC content

Sequence duplication levels

FASTQC metagenomic troubleshoot • 926 views
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How to add images to a Biostars post

I made the changes for you this time so that images are displayed properly. You have to provide the full link to the image.

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And the sequence length distribution error is due to the trimmed sequences won't have the same length as those untrimmed.

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