[HELP] All ASVs occurring only once across samples - 16S amplicon

[Ka1wenSun]

Hi,

Thanks for your help. I'm stuck on this problem for many days... DADA2 finds 8531 ASVs, but all of them occurring only once across samples. I searched past issues, tried use pool = TRUE, but the problem still present. I'm a beginner on coding, couldn't find any problem that explain why.

I don't think there's problem with my data itself, my professor says the readings in data processing looks normal.

[Ka1wenSun]

[Ka1wenSun]

One possible problem is my sequences quality, they has high quality score. Reverse readings have even higher quality than forward.

The laboratory tech who sequenced my samples said it is because they have a new fancy High-Output DNA Sequencing which gives out high quality result.

Because of there are less error in my sample, I couldn't run errR <- learnErrors(filtRs, multithread=TRUE), it will ended up in error code: Error rates could not be estimated (this is usually because of very few reads). So I used errR <- inflateErr(errF, 1) to keep the code running.

[Ka1wenSun]

also when I tried: dada( ... , pool= TRUE), I couldn't run it because the RAM limit (I used my own laptop), I use this code to do it separately. I don't know if this will cause any different.

[Ka1wenSun]

Quick update, I solved the ASVs problem. ASVs occuring only once because the poly-G and poly-C chain at the start and the end of my sequence. By trimming both side, ASVs become shared across samples.