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Forum: Illumina/Solexa 09-14-2018, 03:51 PM
Replies: 3
Views: 316
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, 01:38 AM
Replies: 14
Views: 682
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, 04:35 PM
Replies: 14
Views: 682
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, 03:27 AM
Replies: 14
Views: 682
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, 03:13 AM
Replies: 14
Views: 682
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, 12:17 AM
Replies: 2
Views: 567
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, 01:41 AM
Replies: 3
Views: 758
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, 12:08 AM
Replies: 3
Views: 742
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 07-16-2018, 11:44 PM
Replies: 3
Views: 742
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, 06:26 PM
Replies: 3
Views: 758
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, 12:27 AM
Replies: 2
Views: 496
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, 04:22 PM
Replies: 1
Views: 980
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, 12:12 AM
Replies: 4
Views: 1,391
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, 12:04 AM
Replies: 2
Views: 902
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, 03:43 AM
Replies: 4
Views: 1,391
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...
Forum: Sample Prep / Library Generation 06-25-2018, 12:31 AM
Replies: 3
Views: 998
Posted By nucacidhunter
In Illumina systems fragments up to 1Kb can be...

In Illumina systems fragments up to 1Kb can be clustered and sequenced. But there is a bias toward sequencing smaller fragments. To sequence more large fragments in a library with wide size...
Forum: Sample Prep / Library Generation 06-18-2018, 11:55 PM
Replies: 1
Views: 536
Posted By nucacidhunter
I do not think that there is any hyper sensitive...

I do not think that there is any hyper sensitive method yet to preprare RNA-Seq library from 1 pg RNA.
Forum: Sample Prep / Library Generation 06-18-2018, 11:48 PM
Replies: 8
Views: 1,151
Posted By nucacidhunter
Larger fragment size indicates higher DNA to...

Larger fragment size indicates higher DNA to transposon ratio which could be result of inaccurate pipette or quantification. It does not seem to be result of insufficient mixing.

Reading between...
Forum: Sample Prep / Library Generation 06-17-2018, 11:43 PM
Replies: 8
Views: 1,151
Posted By nucacidhunter
I wonder which SureSelect kit you have used. ...

I wonder which SureSelect kit you have used.

Edit: It seems that you have copied the attached Bianalyzer trace from the SureSelect QXT manual and according to manual it is fine so you can proceed...
Forum: Sample Prep / Library Generation 06-16-2018, 04:23 AM
Replies: 8
Views: 1,151
Posted By nucacidhunter
You can check sheared DNA peak size before...

You can check sheared DNA peak size before starting library prep and shear more if it is too large. Peak should be 150-200 bp. Your Covaris could be faulty.
Forum: Sample Prep / Library Generation 06-15-2018, 05:55 AM
Replies: 8
Views: 1,151
Posted By nucacidhunter
It seems that shearing has not resulted in...

It seems that shearing has not resulted in expected fragment length so library size distribution is too large for SureSelect. Average library insert should be around 150 bp.

It is better to...
Forum: Sample Prep / Library Generation 06-15-2018, 02:28 AM
Replies: 5
Views: 978
Posted By nucacidhunter
You can use Bioanlyser high sensitivity DNA Chip...

You can use Bioanlyser high sensitivity DNA Chip to visualize up to 12Kb and over (minimum 5pg) or gDNA ScreenTape up to 50kb (minimum 1ng). Either of these will indicate the size range suitable for...
Forum: Illumina/Solexa 06-13-2018, 01:10 AM
Replies: 3
Views: 676
Posted By nucacidhunter
Sequence length distribution: it seems to be the...

Sequence length distribution: it seems to be the result of trimming adapters

Duplication Levels: deep sequencing in relation to library diversity

per base sequence content: could be result of...
Forum: Illumina/Solexa 06-13-2018, 12:54 AM
Replies: 4
Views: 573
Posted By nucacidhunter
It should be enough for annealing low diversity...

It should be enough for annealing low diversity short fragments such as adapter oligos. Denaturing should not destroy the DNA as in PCR this happens in every cycle. The fact that your fragments are...
Forum: Illumina/Solexa 06-12-2018, 12:01 AM
Replies: 4
Views: 573
Posted By nucacidhunter
I would suggest to do 1-2 cycle PCR using all...

I would suggest to do 1-2 cycle PCR using all volume of denatured library as template and re-analyse. It is possible that libraries has not re-annealed so is not detectable by the Tape.
...
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