loss of reads after DADA2 as chimeras

User Support
1

Hi,

I am loosing around 70% of my reads as chimeras. Is my truncation parameters right ? or is it due to some other issues?

demux.qzv (291.6 KB)

My parameters were

qiime dada2 denoise-paired \

--i-demultiplexed-seqs analysis/demux-paired-end.qza
--p-trim-left-f 7
--p-trim-left-r 7
--p-trunc-len-f 298
--p-trunc-len-r 256
--o-table table.qza
--o-representative-sequences rep-seqs.qza
--o-denoising-stats denoising-stats.qza

This was the denoising stats

denoising-stats.qzv (1.2 MB)

My sample are faecal pellet samples of rabbits and I have amplified the V3-V4 region of 16S rRNA. Then run them on 600V3 paired end Miseq platform.

Thanks in advance,
Shruthi

2

Hi @Shruthi,
70% is indeed quite a bit, hopefully we can improve on that a bit. Here are some things to consider and follow up questions:

  1. Your trim/truncating parameters look good to me, with plenty of overlap for merging etc. however,
  2. DADA2 can struggle a bit with chimera detection if non-biological sequences are left in your reads, for example if you haven't removed your adapters, barcodes, primers etc. So this is a good starting point to make sure you only have biological sequences in your reads.
  3. On the wet lab side of things, how many PCR cycles did your samples go through? Did you use a high fidelity polymerase? These can often affect chimera production.
  4. Lastly, you can try adjusting the --p-min-fold-parent-over-abundance parameter in dada2.
  5. The good news is that even if for some reason you do have lots chimeras being removed your output is still very good and with the exception of 1/2 samples you could easily just move on and not worry too much about them.
3

Hi @Mehrbod_Estaki,
Thanks for you time in responding to this question.

I did remove my adapters and barcodes in the MiSeq run it self. For wet lab, I used Invitrogen SuperFi master mix for library prep and ran it for 25 cycles in PCR.

I will re-try the run adjusting the parameter you mentioned.

Thanks,
Shruthi