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Forum: Sample Prep / Library Generation 11-16-2018, 10:06 PM
Replies: 4
Views: 228
Posted By nucacidhunter
I am not sure how you have come up with...

I am not sure how you have come up with degradation conclusion. qPCR amplicons goes through excessive cycling (normally 40) and end product can have artifacts and also running them on BA without...
Forum: Sample Prep / Library Generation 11-15-2018, 12:06 AM
Replies: 2
Views: 179
Posted By nucacidhunter
Yes it works and your plan looks fine. Second...

Yes it works and your plan looks fine. Second round of PCR adds the indexes and remaining adapter sequences and is primed from the overhang which is common among amplicons.
Forum: Bioinformatics 11-14-2018, 11:56 PM
Replies: 4
Views: 194
Posted By nucacidhunter
Stretches of G in NextSeq indicates lack of...

Stretches of G in NextSeq indicates lack of signal in those cycles and it could be result of short inserts and adapter-dimer.

In a shot gun library GC content peak will be representative of genome...
Forum: Sample Prep / Library Generation 11-14-2018, 11:46 PM
Replies: 4
Views: 228
Posted By nucacidhunter
Do you mean library degrades after size selection...

Do you mean library degrades after size selection and clean up if you leave the library in fridge or RT?

If the library reagents are contaminated you would not have the library in first place.
Forum: Bioinformatics 11-14-2018, 02:09 AM
Replies: 4
Views: 194
Posted By nucacidhunter
The plots that you are referring should show...

The plots that you are referring should show normal distribution around the GC content of genome in a random library. But IP is not expected to be random so there would be a bias. Extreme GC at the...
Forum: Illumina/Solexa 11-08-2018, 02:47 AM
Replies: 2
Views: 391
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: 324
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: 289
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: 980
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: 980
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: 528
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: 980
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: 447
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: 638
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: 638
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: 717
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: 887
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: 887
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: 887
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: 887
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: 718
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: 1,252
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: 1,182
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: 1,182
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: 1,252
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...
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