Help troubleshoot for improving denoising stats

[Priyanka_Mishra]

In the denoising step using dada2 i have set forward 301 and reverse 260 for truncating. And I got the following denoising stats:

Here the percentage of input passed filter and input merged were too low. So one of my colleague suggested me to take forward 290 and reverse 280 for truncating but still the same trend has been noticed:

I am attaching the quality plot for your reference:
paired-end-demux.qzv (314.8 KB). This plot is before trimming adapters and barcodes.
But after trimming those quality has been improved it seems.
paired-end-demux.qzv (317.2 KB) . So I have used the later one quality plot for denoising step.

So please help us to improve the denoising stats so that we can move ahead for further analysis.
Thank you.

[colinbrislawn (Colin J Brislawn)]

Good morning Priyanka,

Every pipeline is a bit different, but for DADA2, the best way to get more reads to pass the filter is to trim at a shorter length.

This will remove the low quality areas at the end of the read, which I have annotated on you R2 quality graph.

That drop in quality around bp 36 is not helping either.

Another good option is to use only the forward read, as it's quality is excelent!

[Priyanka_Mishra]

So @colinbrislawn could you please suggest how much should I set parameter for trim and truncate length for forward and reverse reads so that I can get input filtered and merged stats to the maximum percentage.
Thank you

[colinbrislawn (Colin J Brislawn)]

I think the reverse read has the lowest quality. So under 260!

How long is the region you amplified with PCR?

[Priyanka_Mishra]

So no need to truncate forward read, only for reverse reads will set truncate 260 right?
V3-V4 region PCR amplified

[colinbrislawn (Colin J Brislawn)]

@Priyanka_Mishra

So no need to truncate forward read, only for reverse reads will set truncate 260 right?

Try it and see!

(I usually try five to ten truncation settings to see what works best. You are off to a great start, so keep going!)

@Priyanka_Mishra

V3-V4 region PCR amplified

Here is how to calculated if the reads will overlap after truncating:

Merging, Quality control and overlapping

Hello Armin, Thank you for posting this really great question along with the detailed screenshots. I think you are on the right track. denoised: 99,046, merged: 57 Yep, that's your problem. Let's fix it! Sure. The pair of Illumina reads sequence your amplicons from both ends, with some overlap in the middle. 250 bp amplicon |-------------------------| 150 bp read |---------------> 150 bp read <---------------| 50 bp overlap (correct!) ^^^^^ Here'…