Single-end approach V3-V4 read length

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Hello qiimer community,

I have some questions about the single-end approach:

  • According to this post my primers that I've used (341F and 805R) flanked a read length of 464bp.

  • Into a single-end approach that I've cut --p-trunc-len = 200 and --p-trim-left = 0, it means that I have a read lenght of 264bp including the V3-V4?

  • This cut may exclude one region V3 or V4? Or my read length keep the region V3-V4?

Thanks in advance!

2

Hello João,

Close! I think these are flipped around a little bit.

my primers that I've used (341F and 805R) flanked a read length of 464bp.

These primers flank an amplicon (PCR product) that 805-341 = 464ish base pairs long. The length of the amplicon varies because the lengths of the gene varies across microbes :microbe:

This is the length of the PCR product in the plate. This is before sequencing, so there's no read yet!

Into a single-end approach that I've cut --p-trunc-len = 200 and --p-trim-left = 0,

This is after sequencing and it's trimming the read. 200-0 = 200 bp long read, which is the first 200 bp of V3. It probably does not reach V4.

Because this cut is done on the computer, the lengths are exact.

Let us know if you have more questions!

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Thanks @colinbrislawn, it's perfectly clear now!

Just one point, from wich read length size (bp) the region sequenced is considered V3-V4 and not just V3?

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341 and 805 are locations on the full length 16S gene.

341F and 805R are locations and directions of the primers, Forward and Reverse

The locations vary depending on the microbe, and how the MSA program works.
Here's one paper as an example: PMC4802574

That one graph shows V4 primers at 515, 519, and 533 so maybe one of those?

Your forward primers is 341F,
the read would need to be 515-341 = 174 bp long to reach 515F
the read would need to be 519-341 = 178 bp long to reach 519R

The core problem is that the location of hypervaiable regions changes between microbes, so these regions are not fixed

See PMC6834636

Notice how these figures both list V1 through V9, but they don't match! :crying_cat_face:

Your questions are excellent because they directly address the messiness of the 16S hypervariable region. You are highlighting what V3-V4 really means, which is complicated!

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