Hi all, I have been working on the metagenomics analysis on a microbial community. It was sequenced on Illumina Nextera sequence, 2x 150bp. I have obtained low map reading quality (properly paired ~23%), and I would like to know what can be done to raise the mapping quality. Here is what I have done:
- Used trimmomatic to trim away low quality reads
- Concatenate forward files into a single forward file (fastq), and the same for reverse files
- FastQC to check quality
- IDBA-UD for assembly (mink = 20, maxk = 100)
- Bowtie2 for mapping, then samtools flagstat for statistics, but obtained low quality mapping (~23%)
The flagstat looks like this:
41641174 + 0 in total (QC-passed reads + QC-failed reads) 0 + 0 secondary 0 + 0 supplementary 0 + 0 duplicates 18804112 + 0 mapped (45.16% : N/A) 41641174 + 0 paired in sequencing 20820587 + 0 read1 20820587 + 0 read2 9847366 + 0 properly paired (23.65% : N/A) 17193728 + 0 with itself and mate mapped 1610384 + 0 singletons (3.87% : N/A) 1906784 + 0 with mate mapped to a different chr 1813704 + 0 with mate mapped to a different chr (mapQ>=5)
I am not sure what caused the low mapping quality, do I need to concatenate files first then use trimmomatic? As this might have caused forward and reverse file losing reads at different position (eg. Forward losing bases at position 1000, but reverse reads losing bases at position 9000).
What could I do to raise the mapping quality?
Cheers and thank