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Forum: Illumina/Solexa 11-25-2019, 09:46 AM
Replies: 5
Views: 433
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, 04:47 PM
Replies: 18
Views: 938
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, 03:00 PM
Replies: 18
Views: 938
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, 02:36 PM
Replies: 18
Views: 938
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, 01:58 PM
Replies: 18
Views: 938
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, 02:06 PM
Replies: 1
Views: 778
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, 06:30 PM
Replies: 5
Views: 1,363
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, 02:06 PM
Replies: 14
Views: 2,762
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, 02:35 PM
Replies: 3
Views: 1,073
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, 09:06 AM
Replies: 1
Views: 691
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, 12:41 PM
Replies: 5
Views: 1,363
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, 12:43 PM
Replies: 5
Views: 1,363
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, 01:04 PM
Replies: 12
Views: 1,519
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, 11:52 AM
Replies: 12
Views: 1,519
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...
Forum: Sample Prep / Library Generation 05-24-2019, 09:23 AM
Replies: 12
Views: 1,519
Posted By itstrieu
I am assuming you are doing the PCR-free...

I am assuming you are doing the PCR-free workflow? What was the input concentration and the concentration after final clean-up. You might have to increase your input concentration or do a PCR step to...
Forum: Illumina/Solexa 03-07-2019, 10:42 AM
Replies: 5
Views: 993
Posted By itstrieu
Which library prep and capture kit are you using?

Which library prep and capture kit are you using?
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