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Search: Posts Made By: nucacidhunter
Forum: General 07-17-2018, 01:41 AM
Replies: 3
Views: 188
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: 149
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: 149
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: 188
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: 231
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: 386
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: 3
Views: 664
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: 489
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: 3
Views: 664
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: 716
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: 295
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: 849
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: 849
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: 849
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: 849
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: 713
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: 484
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: 404
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: 404
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.
...
Forum: Illumina/Solexa 06-08-2018, 05:40 PM
Replies: 2
Views: 333
Posted By nucacidhunter
I have not used these exact primers but in my...

I have not used these exact primers but in my experience two step PCR libraries sequence just fine. Aim for cluster density at 550-750k with 20% PhiX. Read number and variation are similar with qPCR...
Forum: Core Facilities 06-06-2018, 09:01 PM
Replies: 3
Views: 641
Posted By nucacidhunter
This is a rare case and I do not think that...

This is a rare case and I do not think that someone would have any readily available figures. It seems that you want to place your machine in a core lab and operate it yourself. The cost will depend...
Forum: Sample Prep / Library Generation 06-06-2018, 01:09 AM
Replies: 10
Views: 1,268
Posted By nucacidhunter
Relatively short DNA fragments can result from...

Relatively short DNA fragments can result from incomplete DNase activity or degradation during extraction and denatured to ssDNA at elevated temperature used for linearizing RNA. Two possible...
Forum: Illumina/Solexa 05-17-2018, 01:56 AM
Replies: 7
Views: 1,040
Posted By nucacidhunter
If you are going to use commercial full-length Y...

If you are going to use commercial full-length Y adapters or synthesize oligos to make up in house adapters then you do not need to change anything. The reversing P7 and i7 index is required if you...
Forum: Epigenetics 05-16-2018, 02:33 AM
Replies: 2
Views: 488
Posted By nucacidhunter
1- Libraries has been sequenced on a NextSeq flow...

1- Libraries has been sequenced on a NextSeq flow cell that has 4 lanes so the data from lanes for each library needs to be merged into one file for each sample replicates for analysis.

2- You...
Forum: Illumina/Solexa 05-08-2018, 01:40 AM
Replies: 7
Views: 1,040
Posted By nucacidhunter
Following lists some standalone forked (Y)...

Following lists some standalone forked (Y) Illumina style full-length adapters:

GeneRead Adapter set A and B (12 each) from Qiagen
NEXTflex® DNA Barcodes and NEXTflex-HT Barcodes (up to 384) from...
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