Need help with improving the 16S rRNA sequence quality with trimmomatic?

Question

[Kai Xin]

Hi all,

bioinformatic NEWBIE here :D

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

the images below shows the sequence before trimming. However, after trimming the result was about the same. I wonder what parameters should I change to get a better quality sequence.

Also if any of you could explain what are the thresholds that define a good quality sequence that would be great.

Thank you very much! Regards, Kai

[jeevii]

Hello,

just giving a suggestion here to use trim galore tool for data pre-processing [quality and adapter removal]. Have used and got good quality data retained, when applying quality 30 and phred score as 33.

Thanks!