Reverse lanes only?

I am working with multiple lanes of paired-end reads sequenced on an Illumina Genome Analyzer II system. It appears that my forward reads are of much lower quality than my reverse reads (see below). I am trying to denoise my reads in dada2, but when I try to denoise using both the forward and reverse reads, it appears that I am losing A LOT of reads at the "merge" step.

Visualization: paired-end-demux.qzv (285.0 KB)

Paired end Dada2 stats: denoise_1.tsv (1.1 KB)

To me, this seems to mean that the forward and reverse ends are not overlapping. I thought that perhaps I was truncating too much, but this has happened using both of the following codes:

Truncation based on demultiplexed visualization:
qiime dada2 denoise-paired --i-demultiplexed-seqs paired-end-demux.qza --o-representative-sequences rep-seqs-dada2.qza --p-trunc-len-f 45 --p-trunc-len-r 60 --o-table table-dada2.qza --o-denoising-stats stats-dada2.qza --p-n-threads 40

dada2 stats: denoising-stats.qzv (1.2 MB)

No truncation:
qiime dada2 denoise-paired --i-demultiplexed-seqs paired-end-demux.qza --o-representative-sequences rep-seqs-dada2.qza --p-trunc-len-f 0 --p-trunc-len-r 0 --o-table table-dada2.qza --o-denoising-stats stats-dada2.qza --p-n-threads 40

dada2 stats: denoising-stats.qzv (1.2 MB)

I have read about using only the forward reads for an analysis, but is it valid to use only the reverse reads?
I have imported the reverse reads from one lane and denoised, and it appears to be much more successful.
Code:qiime dada2 denoise-single --i-demultiplexed-seqs single-end-demux.qza --o-representative-sequences rep-seqs-dada2.qza --p-trunc-len 0 --o-table table-dada2.qza --o-denoising-stats stats-dada2.qza --p-n-threads 40

Dada2 stats: denoising-stats_SE3.qzv (1.2 MB)

For clarity, each lane contains 30 samples of 16S V3 region and ITS 2 region, and I am using QIIME2-2019.1 on a server.

I am also considering PANDASeq to merge my forward and reverse reads, although I would rather use the Q2 pipeline. Any thoughts?

Thanks in advance,

Hi @LauraMason,
Wow! I haven’t see GAIIx data in a long time! This takes me back to the good old days :older_adult:

It is not because of your parameters, it is because the reads are really really short so I doubt they will overlap at all, unless if you have a very short amplicon (say, 100 nt long).

Yes! Nothing wrong with that. Looks like this is working for you, so I recommend proceeding with that.

Don’t bother. Those forward reads are low quality and even if they were not the read lengths are so short that you could not get anything to merge for ITS2.

Good luck! :shamrock:

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Another question, do you recommend trimming the 5’ end of the reverse reads? It looks like the quality drops off a little bit around 60 bp, but would trimming improve anything?

no don’t bother. the reads are so short and it looks like dada2 yielded enough seqs without trimming.

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