Very low percentage of merged reads in DADA2 with 16S V4, 2×150 bp paired-end sequencing

[SoilRotifer (Mike Robeson)]

Hi @Sergio,

Thanks for the informaiton.

@Sergio

I am also confused about the expected amplicon length for 515F–806R, since I have found values ranging from approximately 250 to 290 bp. With 2×150 bp reads, that would mean approximately 50 bp versus only ~10 bp of theoretical overlap.

The range is often due to including (~290 bp) or not including (~250 bp) the primers as part of the fragment length. That is, standard sequencing approaches sequence through the PCR primers. Meaning the PCR primers appear in the first ~20 bases of your reads at the 5' end. Often users will run cutadapt to explicitly remove these primers prior to downstream processing (search the forum for more threads on this topic).

If the approach you used does in fact sequence through the primers... then unfortunately 2x150bp is generally not long enough to merge the V4 reads. In which case I'd recommend only using the forward reads.

The only way 2x150 works well for the V4 region is if you are using the EMP 16S rRNA gene sequencing protocol, which essentially does not sequence through the PCR primers, thus allowing you to reach further down towards the 3' end, as you are not wasting precious sequencing on the PCR primer for the 2x150 run. See here, for more details.