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Forum: Illumina/Solexa 09-10-2020, 05:57 AM
Replies: 1
Views: 350
Posted By itstrieu
I have sequenced large libraries before and while...

I have sequenced large libraries before and while they do cluster inefficiently, the reads that passed filter are similar to other libraries. Mixing libraries of different sizes can be tricky in...
Forum: Illumina/Solexa 09-08-2020, 05:41 AM
Replies: 8
Views: 2,425
Posted By itstrieu
The first 25 cycles or so is the most important...

The first 25 cycles or so is the most important for library diversity. Was the Phix made fresh for this run or was it from an aliquot? I sometime refreeze denature Phix for later runs and after...
Forum: Illumina/Solexa 09-01-2020, 11:19 AM
Replies: 8
Views: 2,425
Posted By itstrieu
What are you libraries size? I believe PhiX is...

What are you libraries size? I believe PhiX is around 500 bp so smaller libraries will bind more efficiency throwing off your PhiX alignment.

Also we ran a couple of mid output kits and noticed...
Forum: Illumina/Solexa 08-07-2020, 05:47 AM
Replies: 3
Views: 869
Posted By itstrieu
While not asymmetric, we did a 2x300 run on a 500...

While not asymmetric, we did a 2x300 run on a 500 cycle kit by accident and read 1 looked good but read 2 quality dropped after 230 cycles due to reagents starting to run out.

I don't see any...
Forum: Illumina/Solexa 06-22-2020, 06:16 PM
Replies: 7
Views: 1,305
Posted By itstrieu
I am not an informatics person but maybe you...

I am not an informatics person but maybe you could normalize the reads by downsampling. I would run past libraries on the NovaSeq or prep new ones to run on both platforms and compare. It might be...
Forum: Illumina/Solexa 06-22-2020, 01:30 PM
Replies: 7
Views: 1,305
Posted By itstrieu
Oh yea there's definitely a need! Also depending...

Oh yea there's definitely a need! Also depending on how sensitive the experiment is, there is also the issue with index hopping. But I see this only as an issue if you are looking for rare variants.
Forum: Illumina/Solexa 06-22-2020, 12:46 PM
Replies: 7
Views: 1,305
Posted By itstrieu
It makes sense using one NovaSeq kit compared to...

It makes sense using one NovaSeq kit compared to multiple MiSeq kit at that volume. Our break even point is around ~1175 samples on the NovaSeq and TATs becomes an issue for us at that point.

If...
Forum: Illumina/Solexa 06-22-2020, 07:21 AM
Replies: 7
Views: 1,305
Posted By itstrieu
It seems like over kill unless you have enough...

It seems like over kill unless you have enough samples/index to fill out the flow cell. We usually sequence @ 100k reads per 16s sample. I can see it being more cost effective if you are doing...
Forum: Sample Prep / Library Generation 06-10-2020, 07:15 AM
Replies: 1
Views: 1,597
Posted By itstrieu
I don't see why not. I usually spot check with...

I don't see why not. I usually spot check with the bioanalyzer after each PCR step to be sure I get the correct amplicon size.
Forum: Sample Prep / Library Generation 03-31-2020, 05:26 AM
Replies: 12
Views: 2,718
Posted By itstrieu
When we prep 16s libraries, the input is...

When we prep 16s libraries, the input is normalize for 1st PCR. We normalize based on molarity after the clean-up from 2nd PCR using Quant-iT/Qubit and a Bioanalyzer trace. This usually gives us...
Forum: Illumina/Solexa 03-18-2020, 09:08 AM
Replies: 1
Views: 1,259
Posted By itstrieu
Yield is the amount of data (bases) a run...

Yield is the amount of data (bases) a run produced. Usually it is the amount of PF reads x total cycle = yield. You can see the reads that passed filter in the Metrics tab in BaseSpace.
Forum: Illumina/Solexa 11-25-2019, 08:46 AM
Replies: 6
Views: 2,143
Posted By itstrieu
I had a issue with the NextSeq where the Q30...

I had a issue with the NextSeq where the Q30 score for read 2 looked horrible and it was determined to be a pump failure.
Forum: Illumina/Solexa 11-06-2019, 03:47 PM
Replies: 18
Views: 2,312
Posted By itstrieu
If the total reads is 25M under the indexing QC...

If the total reads is 25M under the indexing QC tabs in BaseSpace, it is actually the total PE reads. Under the Metrics tab, READS PF will be half of that. I would ask if they could rerun the library...
Forum: Illumina/Solexa 11-06-2019, 02:00 PM
Replies: 18
Views: 2,312
Posted By itstrieu
You can increase cluster density by increasing...

You can increase cluster density by increasing the loading concentration.

In my post they are paired end reads so double that if you where doing single end reads. Here is a link how many reads...
Forum: Illumina/Solexa 11-06-2019, 01:36 PM
Replies: 18
Views: 2,312
Posted By itstrieu
I would say it is library dependent and what...

I would say it is library dependent and what metrics you are aiming for. For V3V4 sequencing, we use spacer primers to add diversity to the run.

For V4 sequencing, we use 515F (Parada)–806R...
Forum: Illumina/Solexa 11-06-2019, 12:58 PM
Replies: 18
Views: 2,312
Posted By itstrieu
Cluster density is kind of low for a v3 kit even...

Cluster density is kind of low for a v3 kit even for low diversity libraries. We usually sequence the V3V4 region on a V3 600 cycle kit and get around 50M PE reads on ~1000 K/mm2 with around 17 pM...
Forum: Sample Prep / Library Generation 09-04-2019, 01:06 PM
Replies: 1
Views: 1,392
Posted By itstrieu
I have seen vendors recommending qPCR-based assay...

I have seen vendors recommending qPCR-based assay to determine DNA quality prior to library prep for FFPE samples.
...
Forum: Sample Prep / Library Generation 08-28-2019, 05:30 PM
Replies: 6
Views: 2,987
Posted By itstrieu
It was with the MBC. You could increase the...

It was with the MBC. You could increase the post-capture PCR cycle by 1 but you might see higher PCR duplicate reads during analysis. I'm curious to see if there are any differences in the sample...
Forum: Illumina/Solexa 08-23-2019, 01:06 PM
Replies: 14
Views: 3,834
Posted By itstrieu
Our workflow is a little bit different when we...

Our workflow is a little bit different when we use the MiSeq. Usually it is the following:

1. Normalize all samples to 8 nM and pool them together.
2. Qubit the pool to see how far off it is...
Forum: Illumina/Solexa 08-16-2019, 01:35 PM
Replies: 3
Views: 1,536
Posted By itstrieu
I ran into a similar issue recently. If you have...

I ran into a similar issue recently. If you have the Dx version of the NextSeq, there is a software update that can do FASTQ generation on the machine similar to the MiSeq.

The other option is a...
Forum: Sample Prep / Library Generation 08-14-2019, 08:06 AM
Replies: 1
Views: 1,507
Posted By itstrieu
How many cycles did you use for the PCR reaction?...

How many cycles did you use for the PCR reaction? It might be that your primers are exhaust and your PCR products are annealing to each other. See this link:...
Forum: Sample Prep / Library Generation 07-10-2019, 11:41 AM
Replies: 6
Views: 2,987
Posted By itstrieu
SS XT Low Input has a similar workflow to HS....

SS XT Low Input has a similar workflow to HS. After library prep, 1000 ng was inputted into hybridization. Other places where I see can affect yield is lost of the capture beads during the washing...
Forum: Sample Prep / Library Generation 07-09-2019, 11:43 AM
Replies: 6
Views: 2,987
Posted By itstrieu
Which library and capture kit are you using? I...

Which library and capture kit are you using? I tested the Exon V7 probes with SureSelectXT and SureSelectXT HS a while back. XT gave >50 nM while XT HS gave around 10 nM. Started with 200 ng as the...
Forum: Sample Prep / Library Generation 05-24-2019, 12:04 PM
Replies: 12
Views: 2,092
Posted By itstrieu
The only two things I can think of is DNA in...

The only two things I can think of is DNA in buffers containing high concentrations of EDTA, other chelating agents or salts may inhibit the
end repair and A-tailing reaction. The other is something...
Forum: Sample Prep / Library Generation 05-24-2019, 10:52 AM
Replies: 12
Views: 2,092
Posted By itstrieu
Is the workflow ER,AT, Ligation --> 0.8X...

Is the workflow

ER,AT, Ligation --> 0.8X bead cleanup --> Elute in 25 uL follow by Library Amplification --> Post-amplification Cleanup using 1x bead? With the concentration being 100ng/uL, how...
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