[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!

[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.

[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.

[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

[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.

[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.

[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

[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!

[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.

[lac302]

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.

[dannyhi321]

This has not been a complete show stopper for us (as it seems to be for some) so we are watching to see where this ends up.

Illumina must be making a good faith effort to fix this and have just not found a solution yet. If they decide to take this SKU off the market (if it can't be fixed then I don't know what else they can do) that can only lead to more unhappy users than there currently are.

With all due respect, I don't regard the statement "There will be no public announcement until we have 100% identified the problem and removed it" relating to a known problem with a core reagent as acting in good faith. In fact a reagent whose performance metric many users base/based the purchase of their system. Keeping quiet and waiting until a user complains and then reimbursing them is hardly "acting in good faith".

I am glad car manufacturers do not apply the same "logic" to their safety systems!

[GenoMax]

Do you think no feedback re the reagent issue is acceptable?

This has not been a complete show stopper for us (as it seems to be for some) so we are watching to see where this ends up.

Illumina must be making a good faith effort to fix this and have just not found a solution yet. If they decide to take this SKU off the market (if it can't be fixed then I don't know what else they can do) that can only lead to more unhappy users than there currently are.

[dannyhi321]

@dannyhi321: The response you copied above was posted by @James Hadfield earlier (post #37) in this thread. I thought you were asking if there has been any feedback recently and the answer for that is no, AFAIK. Only thing we ended up doing was downgrading MCS to v.2.5.x based on recommendation from Illumina (there is a PQN for that).

We sequence all kinds of libraries (many are low diversity). Some work better than others but we have not had any outright failures. Most of the times the drops in Q-scores have been because of shorter inserts (than expected) otherwise for others we have been able to routinely get Q30 > 75%. We do spike-in 10% or more phiX based on the sample type for low diversity samples.

Do you think no feedback re the reagent issue is acceptable?

[GenoMax]

@dannyhi321: The response you copied above was posted by @James Hadfield earlier (post #37) in this thread. I thought you were asking if there has been any feedback recently and the answer for that is no, AFAIK. Only thing we ended up doing was downgrading MCS to v.2.5.x based on recommendation from Illumina (there is a PQN for that).

We sequence all kinds of libraries (many are low diversity). Some work better than others but we have not had any outright failures. Most of the times the drops in Q-scores have been because of shorter inserts (than expected) otherwise for others we have been able to routinely get Q30 > 75%. We do spike-in 10% or more phiX based on the sample type for low diversity samples.

[dannyhi321]

We are using 2x300 kits. There is no feedback from Illumina. As detailed in this thread, if you feel that you did not get enough data, you can open a ticket with tech support and they may provide a free kit (this may be dependent on having a current maintenance contract).

There IS feedback from illumina:

At Illumina's UK UGM the team discussed the low quality at the end of long-read MiSeq kits. They acknowleged there was an issue and that they were actively working on it. However they have no plans to issue a quality notification until they understand the root cause. This issue has been rumbling on for months, at least a handful of invited users had not even heard of the problem.

Users need to be aware that 600 cycle kits are most likely not to work for long amplicons as the ends of both reads will drop in quality - badly.

The sentiment at the meeting was that issuing a quality notification to make sure customers are aware of this risks damaging confidence in these kits. Whilst many users will be unaffected (small random fragment genomes) or anyone sequencing under 500bp, the kit is clearly not delivering as expected in many long-read situations. Shareholders and investment banks may react negatively to a PQN, but without it too many users are in the dark.

Speak to your FAS and Sales team.

I assume you have not had any issue with the kits ?

Do you feel this is an acceptable response to the ongoing issue?