I have been looking at the first sequencing run of my data which comes from chronic wound infection swabs. The data is Illumina 2x300 paired end reads and samples were prepared following the human microbiome project protocol using primers 515F and 806R. As far as I know there has been no QC performed by the sequencing center but I did receive the reads demultiplexed. From the summary statistics I used the following commands with Dada2:
qiime dada2 denoise-paired --i-demultiplexed-seqs FUMID-demux-1.qza --p-trim-left-f 19 --p-trim-left-r 20 --p-trunc-len-f 290 --p-trunc-len-r 255 --o-table FUMID-table-1.qza --o-representative-sequences FUMID-rep-seqs-1.qza --o-denoising-stats FUMID-denoising-stats-1.qza --p-n-threads 20 --verbose
FUMID-demux-summary-1.qzv (304.9 KB)
From the denoising stats I noticed that I was losing a lot of reads during the removal of chimeras.
Denoising stats pool 1 255bp.tsv (7.7 KB)
Initially I though this might be down to not trimming my reverse reads enough to remove all the poor quality bases. So I repeated the denoising with the following commands:
qiime dada2 denoise-paired --i-demultiplexed-seqs FUMID-demux-1.qza --p- trim-left-f 19 --p-trim-left-r 20 --p-trunc-len-f 290 --p-trunc-len-r 200 --o-table FUMID-table-1.qza --o-representative-sequences FUMID-rep-seqs- 1.qza --o-denoising-stats FUMID-denoising-stats-1.qza --p-n-threads 20 – verbose
And got this out:
Denoising stats pool 1 200bp.tsv (7.7 KB)
So I guess my question is, do you think that I am losing too many reads during the chimera removal stage? If so, is there anything I can do to improve on this?
Let me know if I have left out some vital information