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Forum: Illumina/Solexa 11-08-2018, 02:47 AM
Replies: 2
Views: 334
Posted By nucacidhunter
10 ul with some overage in each well which...

10 ul with some overage in each well which contains pool of two primers.
Forum: Sample Prep / Library Generation 11-02-2018, 12:25 AM
Replies: 3
Views: 256
Posted By nucacidhunter
DNA assays should be fine. I actually keep them...

DNA assays should be fine. I actually keep them in RT and have not seen any adverse affect. Qubit is stable and quarterly calibration is enough.
Forum: Sample Prep / Library Generation 11-01-2018, 02:32 AM
Replies: 4
Views: 215
Posted By nucacidhunter
There is not any practical noticeable difference,...

There is not any practical noticeable difference, though I have not done any systematic comparison.
Forum: Metagenomics 10-17-2018, 12:32 AM
Replies: 5
Views: 860
Posted By nucacidhunter
I do not know why oligo dT has been used in 1st...

I do not know why oligo dT has been used in 1st strand synthesis. It is possible that dA20 oligo has hybridized to oligo T resulting in creating long stretches of T by joining that has been used as...
Forum: Metagenomics 10-16-2018, 01:02 AM
Replies: 5
Views: 860
Posted By nucacidhunter
I do not know how the library has been prepped...

I do not know how the library has been prepped but I notice following:

1- It is stranded (directional) library
2- Probably RNA has been poly adenylated after extraction
3- It contains some short...
Forum: Oxford Nanopore 10-13-2018, 05:00 AM
Replies: 2
Views: 479
Posted By nucacidhunter
There is a discussion thread in Nanopore...

There is a discussion thread in Nanopore community. It can be accessed by logging in and searching for "PromethION barcode".


It has been tried successfully but Nanopre is working on an optimized...
Forum: Metagenomics 10-09-2018, 11:53 PM
Replies: 5
Views: 860
Posted By nucacidhunter
Could you post full sequence of few reads (both...

Could you post full sequence of few reads (both reads if sequenced paired end).
Forum: Illumina/Solexa 09-28-2018, 06:10 AM
Replies: 1
Views: 397
Posted By nucacidhunter
Nextera DNA Flex workflow includes a double SPRI...

Nextera DNA Flex workflow includes a double SPRI size selection. A Pippin size selection will reduce library diversity and is not required unless you have a specific application that needs tight size...
Forum: Sample Prep / Library Generation 09-21-2018, 11:21 PM
Replies: 3
Views: 573
Posted By nucacidhunter
You should not use less DNA. Your thermocycler or...

You should not use less DNA. Your thermocycler or kit also could be faulty (expired, exposed to high temperatures). You can test lower DNA input and see if you get shorter fragments.
Forum: Sample Prep / Library Generation 09-21-2018, 02:13 AM
Replies: 3
Views: 573
Posted By nucacidhunter
The library profile indicates input DNA overload...

The library profile indicates input DNA overload and it can affect on target capture (enrichment efficiency).

Exon length on average are around 200bp and a 1Kb fragment might have hybridized to...
Forum: Illumina/Solexa 09-14-2018, 04:51 PM
Replies: 3
Views: 666
Posted By nucacidhunter
Link to Qubit ssDNA manual below. You might need...

Link to Qubit ssDNA manual below. You might need to use higher volume of pool for quantification. Start with 5 ul and if signal is below detection it can be increased up to 20 ul.
...
Forum: Illumina/Solexa 07-26-2018, 02:38 AM
Replies: 14
Views: 868
Posted By nucacidhunter
Reported density on SAV sometimes could be...

Reported density on SAV sometimes could be incorrect if cluster density is high and software is unable to identify individual clusters. To confirm this is not the case you can examine images of few...
Forum: Illumina/Solexa 07-25-2018, 05:35 PM
Replies: 14
Views: 868
Posted By nucacidhunter
Following usually are the cause of low PF: ...

Following usually are the cause of low PF:
1- Over-clustering
2- Low diversity
3- Sequencing primer quality
4- Adapter and primer quality

I think #4 would be most likely cause in this case if...
Forum: Illumina/Solexa 07-25-2018, 04:27 AM
Replies: 14
Views: 868
Posted By nucacidhunter
Sorry, I meant to ask for %Base in Data by Cycle...

Sorry, I meant to ask for %Base in Data by Cycle plot from SAV.
Forum: Illumina/Solexa 07-25-2018, 04:13 AM
Replies: 14
Views: 868
Posted By nucacidhunter
FastQC report of a bad and good run will be...

FastQC report of a bad and good run will be helpful for troubleshooting.
Forum: Illumina/Solexa 07-25-2018, 01:17 AM
Replies: 2
Views: 698
Posted By nucacidhunter
Fast library degradation is unlikely. If Agilent...

Fast library degradation is unlikely. If Agilent (I assume BA or TapeStation) indicates DNA presence, the issue could be qPCR.
Forum: General 07-17-2018, 02:41 AM
Replies: 3
Views: 1,212
Posted By nucacidhunter
Yes, specially if you are using proof reading...

Yes, specially if you are using proof reading polymerase. The primers also will be too long and would need gel or HPLC purification which will increase costs.

It will be more cost effective if...
Forum: General 07-17-2018, 01:08 AM
Replies: 3
Views: 1,146
Posted By nucacidhunter
Using indexes would give more options for...

Using indexes would give more options for multiplex sequencing with no adverse effect.
Forum: General 07-17-2018, 12:44 AM
Replies: 3
Views: 1,146
Posted By nucacidhunter
Barcoding in this context is vague. I wonder if...

Barcoding in this context is vague. I wonder if you mean using indexed adapters for multiplex sequencing.
Forum: General 07-16-2018, 07:26 PM
Replies: 3
Views: 1,212
Posted By nucacidhunter
This is to reduce the number of tags and hence...

This is to reduce the number of tags and hence sequencing requirement. Sequencing is much cheaper than 5 years ago and may not be a factor in experiment design.

One need to estimate tag number if...
Forum: Illumina/Solexa 07-09-2018, 01:27 AM
Replies: 2
Views: 562
Posted By nucacidhunter
If they have been sequenced on MiSeq, you could...

If they have been sequenced on MiSeq, you could have cross contamination among i7 primers.

1- It could have happen during primer synthesis where yuo will get more of other i7 index from a...
Forum: General 07-07-2018, 05:22 PM
Replies: 1
Views: 1,445
Posted By nucacidhunter
It should not be an issue as it requires polyA...

It should not be an issue as it requires polyA region for binding and priming. If there are such sequences (polyT) throughout the exons then some 1st strand cDNA might be primed but it will have...
Forum: General 07-05-2018, 01:12 AM
Replies: 4
Views: 1,815
Posted By nucacidhunter
In theory TruSeq can be used but I have not seen...

In theory TruSeq can be used but I have not seen any convincing evidence from Illumina using this kit for FFPE RNA-Seq. Instead, they have focused more on targeted approaches such as RNA exome...
Forum: Pacific Biosciences 07-04-2018, 01:04 AM
Replies: 2
Views: 1,210
Posted By nucacidhunter
For PacBio you can use different quantity and...

For PacBio you can use different quantity and pool based on molar concentration (after ligation step) to obtain relatively equal read coverage.

Points to consider:
1- adding twice the required...
Forum: General 06-27-2018, 04:43 AM
Replies: 4
Views: 1,815
Posted By nucacidhunter
You have enough quantity and relatively good...

You have enough quantity and relatively good quality RNA for a FFPE sample. Ribo-depletion will be good choice as it is open for transcriptome wide discovery and sequencing cost with NovaSeq is also...
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