Illumina sequencing - paired end reads

Question

[nell]

Hi,

I have R1 and R2 paired-end reads from illumina sequencing (2x300 pb, objective / target: region V4 with: forward primer: 515-533 reverse primer: 787-806).

Our observed case :

"normal"/ expected Case :

Is our case possible? Is it a problem of sequencing?

Thank you.

[Mask]

From where did you get the data? From a public database or your own custom data? If from public database share the identifier

[nell]

It's our own custom data, and not from public database.

[Nicolas Rosewick]

all of your R1 and R2 reads have this type of orientation ? or only a subset ?

[nell]

Yes, all of R1 and R2 reads have this type of orientation.

[WouterDeCoster]

Were those reads trimmed?

[nell]

No, those reads weren't trimmed.

[nell]

I think of having found the explanation here: http://thegenomefactory.blogspot.fr/2013/08/paired-end-read-confusion-library.html

(See "Adaptor read-through")

[Devon Ryan]

Are you sure you didn't just swap the R1 and R2 files?

[Gabriel R.]

Did you do a gel cut? When you did, were your adapter already added in the mixture? If so, you probably selected for very short fragments.

Answer 1

[h.mon]

Looking at your outermost primer positions, the expected amplicon has 291 bp. With 300 bp paired sequencing, it seems to me what you are observing is the expected.

[Brian Bushnell]

Adding to that - 806-515=291. You can't expect to see longer inserts than that, so you should expect all of your reads to have insert sizes shorter than read length and thus run into adapter sequence at the tails. That said, hypervariable regions are called that for a reason, so potentially some pairs may have an insert size over 300bp. It's a good idea to use a read merger that can handle both scenarios (e.g., not FLASH). Or, adapter-trim the reads prior to merging, which is a good idea regardless.

[Gabriel R.]

A brief comment, we published leeHom, a tool for ancient DNA but suitable for such situations. The probability of seeing the adapter and the sequence overlap are combined in a single probabilistic model and maximum-likelihood is used to produce the most likely intervening DNA sequencing. So it combines the adapter trimming/merging for sequence of arbitrary length.

Renaud, Gabriel, Udo Stenzel, and Janet Kelso. "leeHom: adaptor trimming and merging for Illumina sequencing reads." Nucleic Acids Research 42.18 (2014): e141-e141. https://grenaud.github.io/leeHom/