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  • Shotgun Meta of Environ Sam: Per Base Seq Cont Per Seq GC Cont failed aft trimming

    Dear all,

    I am really a newbie for analyzing shotgun metagenomics data. Here I encountered some issues when I checked the quality of my data. I post my concerns here and hope someone can help me.

    DNA samples: Genomic DNA isolated from environmental samples (soil, sewage, or freshwater). We are interested in the community structures of bacteria and archaea in those samples as well as detecting functional genes.

    Sequencing platform: Illumina, Shallow Metagenomics, Shotgun sequencing of DNA, Paired-end sequencing

    Library: Nextera kits (I got this information when running TrimGalore!)

    Concern-1: Per Base Sequence Content
    Before trimming, I checked the quality of the raw data using FastQC + MultiQC. Many samples failed the Per Base Sequence Content test with biased composition at the 5-end (see the attached Per Base Sequence Content-No trimming.jpg), and all samples failed the Adapter Content test (see the attached Adapter Content--No trimming.jpg). I then thought that I needed to trim the 5-end by removing 15 bp from each read and also trim the adapters. I trimmed all the raw reads with TrimGalore! with the following command:
    ===============
    ~/TrimGalore-0.6.5/trim_galore --clip_R1 15 --clip_R2 15 --paired read_1_sample_1.fastq.gz read_2_sample_2.fastq.gz read_1_sample_2.fastq.gz read_2_sample_32.fastq.gz … read_1_sample_N.fastq.gz read_2_sample_N.fastq.gz
    ===============
    After the trimming, I ran FastQC + MultiQC and found that, surprisingly, all samples failed the Per Base Sequence Content test. I found that all samples shared the same pattern: the 3-end is significantly biased with the content of C being very low (see the attached Per Base Sequence Content-After trimming.jpg).
    My question is, should I worry about the bias at the 3-end? Or, should I further trim the 3-end? Specifically, the curve/line for C is roughly horizontal before the trimming. Why this curve/line dropped to almost zero after the trimming? An online discussion (https://github.com/FelixKrueger/Trim...-auto-detectio) mentioned that [Note that the sharp decrease of A at the last position is a result of removing the adapter sequence very stringently, i.e. even a single trailing A at the end is removed.] However, as far as I can understand, the trimming at the 3-end just means removing the sequencing of the adapter (if there is sequencing read-through). The trimming should not affect the remaining (i.e., the sequence that is kept) sequences. If the curve of C before the trimming is horizontal, it should also be horizontal after the trimming. I am a bit confused.

    Concern-2: Per Sequence GC Content
    Before trimming, I found that many samples failed the Per Sequence GC Content test because of the multiple peaks in the plot (see the attached Per Sequence GC Content--No trimming.jpg). I thought that this failure was due to adapter contamination. However, after trimming, many samples still have the issue (see the attached Per Sequence GC Content--After trimming.jpg).

    My question is, why my samples show multiple peaks? Is it possible that my samples contain more than one dominant species? Or, the multiple peaks were due to sequencing/process errors? How should I fix this issue?

    Question-3: The sequencing I did is shallow sequencing. Also, my samples are not pure culture samples--they contain millions of different species of microbes. We will examine the microbial community structure and detect/find functional genes. In this case, should I do assembly before the downstream analysis? I read some online discussions. Some suggests assembly, and some says that it is better to skip the assembly. I am really new in this area and do not know which (with vs. without assembly) is a better choice.

    Thanks for reading this posting!
    Attached Files

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