Hi qiime2 team. I am trying to use dada2 on my 16S paired-end sequences, from a 2 x 250 bp illumina run. But I am losing ~50% of my reads at the merging step. This is despite the fact that there should be enough overlap. My reads contain primers. I have tried both: removing the primers with cutada…

Hi @Jess_Mackay , Could you share the command you're using, please, with all your parameters? Best, Justine

Sorry, I forgot to include that. Cutadapt: qiime cutadapt trim-paired --i-demultiplexed-sequences demux.qza --p-front-f GTGYCAGCMGCCGCGGTAA --p-front-r CCGYCAATTYMTTTRAGTTT --o-trimmed-sequences demux-trimmed.qza Dada2: qiime dada2 denoise-paired --i-demultiplexed-seqs demux-trimmed.qza …

Hi @Jess_Mackay , Thanks for the commands! [image] Jess_Mackay: I am trying to use dada2 on my 16S paired-end sequences, from a 2 x 250 bp Illumina run. But I am losing ~50% of my reads at the merging step. This is despite the fact that there should be enough overlap. [image] Jess_Mackay: …

Thanks for your reply @jwdebelius [image] Jess_Mackay: This may be because when Dada2 denoises, it trims before it denoises. I'm not sure, but it's possible that your reads are getting trimmed at the denoising stage and then they're failing to merge. I am a bit confused what you mean here. D…

Hi Jess, [image] Jess_Mackay: Are you saying that it is possible that the denoising is then leading to issues merging?? I am very new to this so still learning. Yes, that's what I'm saying. (It may be the quality filtering step, rather than the denoising). But, essentially in one of these st…

I'm not sure about this. I know that the --p-trunc-q parameter (with a default of 2) truncates reads with low quality sequences. But it says in the documentation that "If the resulting read is then shorter than 'trunc-len-f' or 'trunc-len-r' (depending on the direction of the read) it is discarded."…

Hi @Jess_Mackay , Just wanted to highlight something here. [image] Jess_Mackay: and there should still be plenty of overlap so this shouldn’t cause problems merging. Not quite true in this case. With your primer set we would expect an amplicon size of 926-515= ~414 bps. Given your 2x250 ru…

Hi @Mehrbod_Estaki Thanks for replying to my previous post ( Dada2: losing reads during merging - #10 by Jess_Mackay the topic got closed because I took too long to reply, so am starting a new topic). I appreciate your response but I am unsure if it is correct. You said: With your primer set we w…

(I've reopened this issue and merged back in the thread!) (Now I'll let the excellent Mehrbod answer your question.)

Thanks @colinbrislawn ! Hi @Jess_Mackay , You are right, I didn't realize the run being referred to was the one you had removed the primers using cutadapt first. I just looked at the quality plots which had shown 2x250 run (usually means primers haven't been removed). So you are correct that your me…

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