Hi all,
I have just started to learn about bioinformatics and I need help with it.
I have enriched some microbes from wastewater anaerobic sludge and sent them for 16S rRNA sequencing.
Based on the QC result I got after running trimmomatic, I am still not able to get a good quality sequence. The following is the code I ran for trimmomatic. Can you all help me with this?
trimmomatic PE -threads 2 -phred33 \
Raw160823_1.fastq.gz Raw160823_2.fastq.gz \
Raw160823_1P.fastq.gz Raw160823_1F.fastq.gz Raw160823_2P.fastq.gz Raw160823_2F.fastq.gz \
HEADCROP:10 SLIDINGWINDOW:4:30 MINLEN:50
Thank you very much!
Regards,
Kai
@Kai Xin Chia I suggest you to set the parameters as default in trimmomatic and run to see any diffrences
Sequence length distribution changes after trimming if you have unrelated sequences in your data. Since, in most cases untrimmed lemgth was set to 150bp.
If default parameters do not work, you may have to tweak the parameters of the software, such as choosing right adapter, seq length to keep and so on
I would be careful about trimming depending on how your primers were setup. As it is based on short-read Illumina technology, you will have primers tiling the 16S V1-V8 regions. If you trim towards the 3' end, you may find that you might not be able to OTU clustering. I've had a customer run 150x2 protocol when they should've run 300x2 protocol for QIAGEN's 16S/ITS kit and the OTU clustering didn't work on the 150x2 protocol (whereas the protocol stated 300x2). But perhaps trimming a dozen or so bases wouldn't cause issues, worth a try!
you can use cutadapt or prinseqlite in bash terminal, these are having quality trimming options.
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