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Forum: Illumina/Solexa Today, 12:03 AM
Replies: 3
Views: 90
Posted By nucacidhunter
Possible important issues: 1- degraded or...

Possible important issues:

1- degraded or low concentration of blocking oligos
2- inefficient hybridisation step (temperature, time, reaction composition)
2- non-stringent wash steps
Forum: Illumina/Solexa Yesterday, 11:58 PM
Replies: 1
Views: 77
Posted By nucacidhunter
High value for pre-phasing could be symptom...

High value for pre-phasing could be symptom rather than the cause. More info on phasing:
...
Forum: Illumina/Solexa Yesterday, 11:39 PM
Replies: 3
Views: 90
Posted By nucacidhunter
It indicates that on target reads (enrichment) is...

It indicates that on target reads (enrichment) is low and most of reads are from background. This figure for most kits is above 70%. At least one step in target capture has been non-optimal.

I...
Forum: Sample Prep / Library Generation 12-16-2018, 12:00 AM
Replies: 1
Views: 131
Posted By nucacidhunter
Double SPRI size selection window will be much...

Double SPRI size selection window will be much larger than 50 bp. For your target you need to use Sage Pippin or gel extraction.
Forum: Sample Prep / Library Generation 12-15-2018, 02:39 AM
Replies: 4
Views: 156
Posted By nucacidhunter
Bead clean up with 0.8x should remove it. It may...

Bead clean up with 0.8x should remove it. It may be necessary to do two clean ups for complete removal. Clean ups should be done with all samples that will be compared if the aim is DGE analysis.
Forum: Sample Prep / Library Generation 12-15-2018, 02:33 AM
Replies: 1
Views: 92
Posted By nucacidhunter
For PCR based amplification of 16S variable...

For PCR based amplification of 16S variable regions RNase treatment usually is not necessary. It will impact correct quantification by spectrophotometric methods if it is included in the workflow. If...
Forum: Illumina/Solexa 12-14-2018, 12:33 AM
Replies: 18
Views: 6,223
Posted By nucacidhunter
Heating denatures the oligo self-dimers and...

Heating denatures the oligo self-dimers and possible secondary structure. Skipping heating will reduce the available adapter for ligation. Its impact will depend on ratio of adapter to template RNA....
Forum: Bioinformatics 11-27-2018, 10:22 PM
Replies: 2
Views: 341
Posted By nucacidhunter
As SNPsaurus has mentioned it is more likely...

As SNPsaurus has mentioned it is more likely library prep issue. Posting FastQC output for raw data would be more informative.
Forum: Sample Prep / Library Generation 11-21-2018, 11:08 PM
Replies: 4
Views: 346
Posted By nucacidhunter
It will damage but I am not sure to what extent....

It will damage but I am not sure to what extent. You can work around it by staining and marking target sizes on the marker lane cut from the gel and using it as a guide for cutting target region from...
Forum: Sample Prep / Library Generation 11-16-2018, 10:06 PM
Replies: 4
Views: 416
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: 348
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: 378
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: 416
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: 378
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: 543
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: 482
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: 448
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: 1,405
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: 1,405
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: 647
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: 1,405
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: 543
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: 763
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: 763
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: 789
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.
...
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