[DRYTCYV]

My last run worked fine even overclustered at 1800k/mm2, 85%PF.

I got one of that cartridges

Our FAS said that Illumina wants to release "the new" V3 kit after Summer.
The last V3 kit we used as a V2 2x250 cycles, the difference is that the V3 got more tiles, so, more data even with the 2x250.

V2 kit its the best kit, in terms of quality and less sensible to overclustering. If Illumina change the V2 kit tiles from 28 to 38 tiles, it would be the best of both worlds.

[dannyhi321]

Is this only an issue for amplicon sequencing? I haven't run a V3 600 cycle kit in a few months but have a library to sequence next week...WGS, FastQ only.

My last run worked fine even overclustered at 1800k/mm2, 85%PF.

I typically run the 600 cycle kits at 2x275. Quality over quantity is fine for my needs.

I think you will find your logic is unsound, a 2x300 kit that only "works" at 2x275 "sometimes" is not quality!

Do you think you could share the Q Score graphs? would be interesting!

[Shimul]

Hello there,

I am running my bacterial library using Miseq V3. The Q30 is now 83.1% after 250 cycles, yesterday it was 96% after 70 cycles. But rest of the parameters are consistent: clusters PF 91.4%, cluster density 1092K/mm^2, which are the same as yesterday. Do you speculate anything? DO you think that the run will be still okay?

Many thanks.

Shimul

[Brian Bushnell]

Illumina's "Q30" values are completely fabricated; I suggest you ignore them. If you want to know the actual quality of your reads, you need to map them. For example:

bbmap.sh in=reads.fq ref=ref.fa mhist=mhist.txt qhist=qhist.txt qahist=qahist.txt

This will give you histograms of the actual quality of your reads.

[nucacidhunter]

Hello there,

I am running my bacterial library using Miseq V3. The Q30 is now 83.1% after 250 cycles, yesterday it was 96% after 70 cycles. But rest of the parameters are consistent: clusters PF 91.4%, cluster density 1092K/mm^2, which are the same as yesterday. Do you speculate anything? DO you think that the run will be still okay?

Many thanks.

Shimul

Run is fine and you would expect Q30 drop a bit more as cycle number increases. Read 2 quality will be lower than read 1 as well. Cluster density and PF% is calculated early in sequencing cycles and will not change.

[jlli2000]

Mixed different 16S metagenomics run

Does anyone have the experience of mixing different 16S 806R , 16S 926R and 18S libraries into one MiSeq run? How about mixing 16S onestep PCR libraries with two step PCR libraries?

Thanks,

Jin

[d00b]

you can mix multiple amplicons

Hi Jin,

we do run multiple amplicons that labeled with different index primers.
However, we do always see index contamination in very low level.

We do two step library preparation.
1) amplification with Miseq adapter
2) Index amplification with different index combination

pool library based on realtime PCR result.

[thermophile]

The biggest issue when mixing amplicons is the lenghts. I've successfully mixed 16s v4 (~300bp) with amplicons that are as small as 190bp and as large as 450 and run on 2x250. Your q30 plot will look weird, but I haven't had any of these mixed runs fail in a way that seems dependent on the mixing. Remember to calculate nM for each length and that the shorter frags will bind better than the longer ones.

[Jessica_L]

My lab is looking at bringing in the TruSight Tumor 15 assay. It seems like most of the v3 issues are related to low complexity libraries (16s RNA-seq reads, etc.) so I'm assuming that we'll be okay with the TST15? I'm wondering if we'll run into problems with some of our other methods, though. We also run the Archer Dx FusionPlex for RNA Fusion detection, and the molecular barcodes in assay have a low complexity region in read 1.

Does anyone have any experience with TST15 or other assays (aside from 16s rRNA) on the v3 chemistry?

Thanks!