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Search: Posts Made By: nucacidhunter
Forum: Sample Prep / Library Generation Yesterday, 11:55 PM
Replies: 1
Views: 99
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 Yesterday, 11:48 PM
Replies: 7
Views: 423
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: 7
Views: 423
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: 7
Views: 423
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: 7
Views: 423
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: 4
Views: 284
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: 282
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: 227
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: 227
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: 164
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: 255
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: 1,095
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: 820
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: 376
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: 820
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...
Forum: Illumina/Solexa 05-07-2018, 03:58 AM
Replies: 7
Views: 820
Posted By nucacidhunter
You can buy adapters from several vendors...

You can buy adapters from several vendors (Illumina, Bioo Scientific, Roche and others) with up to 384 index without buying a full kit.
Forum: Illumina/Solexa 04-25-2018, 04:46 AM
Replies: 6
Views: 679
Posted By nucacidhunter
NEB recently released unique duel index primers...

NEB recently released unique duel index primers and it should be easier to use them with NEB Illumina libray prep kit.
Forum: Illumina/Solexa 04-25-2018, 04:09 AM
Replies: 6
Views: 679
Posted By nucacidhunter
There are commercially available unique duel...

There are commercially available unique duel indices from venders such as Illumina, NEB, Bioo Scientific, NuGEN (up to 96 UDI) and Integrated DNA technologies (up to 384). These are offered as...
Forum: Bioinformatics 04-22-2018, 03:14 AM
Replies: 13
Views: 1,412
Posted By nucacidhunter
This is because ONT sells their consumables ...

This is because ONT sells their consumables directly but other companies such as PacBio sell them through a local distributor that have different margin and shipping costs depending on global...
Forum: Illumina/Solexa 04-19-2018, 02:20 PM
Replies: 7
Views: 697
Posted By nucacidhunter
I should correct that by fragment numbers I meant...

I should correct that by fragment numbers I meant fragments that are flanked by both RE and are in size selection window. With a 4x6 cutter in a 30 Gb genome I guess there will a lot more restriction...
Forum: General 04-19-2018, 02:10 PM
Replies: 3
Views: 430
Posted By nucacidhunter
GBS is a common name used for even...

GBS is a common name used for even non-restriction enzyme based methods. For instance, Illumina recently released a GBS kit that prepares libraries without RE o. ddRAD generally results in good...
Forum: Illumina/Solexa 04-19-2018, 03:18 AM
Replies: 7
Views: 697
Posted By nucacidhunter
First of all standard RRBS using MspI may not be...

First of all standard RRBS using MspI may not be a good approach for plants as RRBS interrogates methylation status of C in CpG context which is most relevant for mammalians.

In a pilot RRBS you...
Forum: General 04-19-2018, 02:42 AM
Replies: 3
Views: 430
Posted By nucacidhunter
Methylation sensitive enzymes are more useful for...

Methylation sensitive enzymes are more useful for plants as their genome contains large repetitive regions due to duplication events. PstI is methylation sensitive in plants as in plants methylation...
Forum: Illumina/Solexa 04-04-2018, 01:47 PM
Replies: 13
Views: 1,016
Posted By nucacidhunter
Good points have been raised in posts above. A...

Good points have been raised in posts above. A run with 40% PhiX spike in should clear whether your library has any issues or if fail is due to preparation and sequencing set up.
Forum: Illumina/Solexa 04-04-2018, 03:15 AM
Replies: 13
Views: 1,016
Posted By nucacidhunter
if I understand correctly, individual libraries...

if I understand correctly, individual libraries BA profile was fine but the library pool did not have any DNA according to Qubit, BA and qPCR?

If library PCR amplification post adapter ligation...
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