Hello all! Setup: I have 16S V3-V4 amplicon sequences using 341F–805R primers. Samples are extracted from soil. The raw data have barcodes and primers, so I use cutadapt to remove it. Here is the data after barcodes and primers removal: trimmed-seqs.qzv (321.6 KB) Problem: When I run DADA2, I …

Hi timanix, I checked other forum post, if I run it with 230 for both sides, I think I will have 4bp gap, right? (forward read) + (reverse read) - (length of amplicon) - = overlap 230+230-464 = -4 bp, then my reads cannot merge at all because they don't overlap. [image] Mergin…

[image] Xinming_Xu: Or, should I discard truncation and lower the value of overlap region which may save the reads loosing on merging step? In that case, probably it is an optimal solution! Decreasing minimum overlap with disabled truncation may save some reads :clap:.

Hi! Sorry for bothering you again! I tried to use the minimum overlap parameter, I set --p-min-overlap 4, actually the result doesn't change too much. It is basically the same as the no truncation result. Seems I can't do anything else to improve my results? Do you have any suggestions? Thanks!

Hi! I am running out of ideas :thinking: Based on your results, you are still loosing reads on merging step after filtering. From one side, you have enough reads to proceed, from another - you results may be biased towards bacteria with shorter amplicons. So option 1 will be just proceed with dis…

Hi @Xinming_Xu ! Got a hint that you also may take a look on Figaro tool (not in Qiime2) that may help determine best truncation parameters.

Hi, really appreciate for all your suggestion! I've seen people using figaro, so first I give a shot to this method. So, the recommended forward truncation position is 238 and the recommended reverse truncation position is 249. [ { "trimPosition": [ 238, 249 …

Hi @Xinming_Xu So I will suggest to proceed with option 2 or try option 3 to see if it will improve amount of reads.

Hi, I've had a similar problem with some soil DNA. What I found worked best was to use only FWD reads and adjust the p=min to 4. Then I was able to keep more than 70% of reads after DADA2, whereas before adjusting I was only keeping between 30-40%. Interestingly, this is only happening for certain s…

Hi, Thanks for your suggestion, I used V3V4, 341F-804R region, and the forward/reverse reads are around 245bp. So it's because the primer sets using or it's soil DNA caused only FWD reads are the best case you've got? I used powersoil kit (Qiagen) to extract my DNA.

I have used both the PowerSoil and a phenol:chloroform extraction method - the PowerSoil does seem to give me better quality DNA, especially after I add some powdered skim milk to the bead-beating step. I'm sequencing reads on a MiSeq v3 600, also with the 341F-804R primer pairs. The extractions I p…

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DADA2: loss too many reads after filtering

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