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Forum: Sample Prep / Library Generation 12-06-2017, 03:02 PM
Replies: 5
Views: 332
Posted By nucacidhunter
150 bp is the target and it looks fine. The...

150 bp is the target and it looks fine. The larger peaks depend on presence of other RNA species and would not be a concern. You might be able to correlate them with input RNA peaks if you have run a...
Forum: General 12-06-2017, 02:32 AM
Replies: 6
Views: 392
Posted By nucacidhunter
You need to walk out of region as far as you can...

You need to walk out of region as far as you can get a good priming site and it does not have to be long.
Forum: General 12-05-2017, 12:54 AM
Replies: 6
Views: 392
Posted By nucacidhunter
I do not know about specifics of your target...

I do not know about specifics of your target regions. To be more successful you can try different primer sets around your region of interest and also consider a nested or semi-nested approach.
Forum: Illumina/Solexa 12-04-2017, 07:12 PM
Replies: 4
Views: 401
Posted By nucacidhunter
It contains 0.1 mM EDTA in comparison to TE with...

It contains 0.1 mM EDTA in comparison to TE with 1 mM EDTA.
Forum: Illumina/Solexa 12-03-2017, 11:58 PM
Replies: 4
Views: 401
Posted By nucacidhunter
If you are not doing bead normalisation step, any...

If you are not doing bead normalisation step, any common buffer such as TE, TE0.1 or EB will do it. For bead normalisation, EB will be better choice as its pH (8.0) is the same as RSB (RSB is...
Forum: Sample Prep / Library Generation 11-21-2017, 11:20 PM
Replies: 252
Views: 84,989
Posted By nucacidhunter
Look at this RNAseH based method:...

Look at this RNAseH based method: http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0042882#s4
Forum: Sample Prep / Library Generation 11-09-2017, 12:16 PM
Replies: 9
Views: 669
Posted By nucacidhunter
Using a 4 and 6 cutter combination and 100 bp...

Using a 4 and 6 cutter combination and 100 bp window cut should give over 10k tags. I would suggest to use 5 ng of digested-ligated DNA in 10-12 PCR cycle and check the size distribution. The profile...
Forum: RNA Sequencing 11-09-2017, 02:16 AM
Replies: 5
Views: 680
Posted By nucacidhunter
It seems that duel index adapters has been used...

It seems that duel index adapters has been used and clean up has been inefficient in removing excess adapters. The best way will be to tackle it after adapter ligation by reducing clean up bead...
Forum: Illumina/Solexa 11-09-2017, 01:53 AM
Replies: 15
Views: 928
Posted By nucacidhunter
I guess you mean 500 pg and that should be fine....

I guess you mean 500 pg and that should be fine. With your 16kb target and 5000x coverage you require 80Mb sequencing per sample. The required read number (2x150) would be ~267k plus some reads to...
Forum: Illumina/Solexa 11-08-2017, 06:32 PM
Replies: 15
Views: 928
Posted By nucacidhunter
Spiky pattern is good indicator of ow diversity. ...

Spiky pattern is good indicator of ow diversity.

Your samples if tagmented randomly would produce around 15,000x15,000 fragments so it is not low diversity. PhiX is only 5.4 kb.

This library...
Forum: Sample Prep / Library Generation 11-08-2017, 01:31 PM
Replies: 9
Views: 669
Posted By nucacidhunter
You can add more RE provided that the final...

You can add more RE provided that the final combined volume does not exceed 10%. It is better to do a test digest on MluCI to rule out bad batch of RE.

The issue mentioned by SNPsaurus could be...
Forum: Illumina/Solexa 11-08-2017, 12:21 AM
Replies: 15
Views: 928
Posted By nucacidhunter
I think that A is better because the other two...

I think that A is better because the other two show signs of low diversity library. For optimum read number you would need to quantify the library with qPCR and take the average size of area under...
Forum: Illumina/Solexa 11-07-2017, 06:19 PM
Replies: 15
Views: 928
Posted By nucacidhunter
Would you elaborate on the experiment aim because...

Would you elaborate on the experiment aim because interpretation would depend on the final aim.

Edit: Are these post PCR libraries cleaned with beads?
Forum: Illumina/Solexa 11-07-2017, 02:08 AM
Replies: 13
Views: 868
Posted By nucacidhunter
Two issues: 1- low sequence diversity at the...

Two issues:
1- low sequence diversity at the start which has resulted in low PF%. This should not happen if following standard RAD-seq protocol

2- short inserts and adapter-dimers that start...
Forum: Sample Prep / Library Generation 11-06-2017, 03:32 PM
Replies: 9
Views: 669
Posted By nucacidhunter
A single digest is far better than sequential...

A single digest is far better than sequential digest if both enzymes are active in the same temperature and buffer (yours are).

In ideal reaction condition incomplete digestion should be minimal....
Forum: Illumina/Solexa 11-02-2017, 12:11 AM
Replies: 13
Views: 868
Posted By nucacidhunter
I wonder if you can post %Base from Data by cycle...

I wonder if you can post %Base from Data by cycle pan.
Forum: Bioinformatics 10-30-2017, 02:20 AM
Replies: 5
Views: 507
Posted By nucacidhunter
You have not given much details but one reason...

You have not given much details but one reason would be sequencing configuration for example, R1 was set to less cycles than R2.
Forum: Sample Prep / Library Generation 10-27-2017, 06:12 PM
Replies: 2
Views: 592
Posted By nucacidhunter
I would trust the size indicated by gel. Three...

I would trust the size indicated by gel. Three issues with Tape:

1- Tape has expired and sometimes that will affect the sizing accuracy
2- The used Tape (HS D5000) accuracy for sizing short...
Forum: Illumina/Solexa 10-24-2017, 04:25 PM
Replies: 13
Views: 743
Posted By nucacidhunter
Easiest way will be to ask client the kit they...

Easiest way will be to ask client the kit they have used and re-demultiplex with a new sample sheet listing all indices available for the kit. Also, if it is a duel index it might be that index2...
Forum: Illumina/Solexa 10-19-2017, 02:59 PM
Replies: 2
Views: 431
Posted By nucacidhunter
And here is the link for Nextera low plex pooling...

And here is the link for Nextera low plex pooling guide:

https://www.illumina.com/documents/products/technotes/technote_nextera_low_plex_pooling_guidelines.pdf
Forum: Illumina/Solexa 10-19-2017, 11:33 AM
Replies: 5
Views: 508
Posted By nucacidhunter
I am not sure if Nextera preference site has been...

I am not sure if Nextera preference site has been studied in details. It just was a precautionary note and in most experiments it does not affect final results.

Nextera works on eukaryotic DNA as...
Forum: Illumina/Solexa 10-19-2017, 03:55 AM
Replies: 5
Views: 508
Posted By nucacidhunter
Generally tagmentation will work with any dsDNA....

Generally tagmentation will work with any dsDNA. Two points to note:
1- Some sequences from both 3' and 5' end will not be sequenced because transposon requires some region to engage with DNA. It is...
Forum: Illumina/Solexa 10-18-2017, 10:28 PM
Replies: 8
Views: 1,076
Posted By nucacidhunter
Plus 7 dark cycles used for index2 read so 85...

Plus 7 dark cycles used for index2 read so 85 cycles in total. This seems to be 10 cycles more than Illumina specification.
Forum: Illumina/Solexa 10-18-2017, 01:16 PM
Replies: 5
Views: 508
Posted By nucacidhunter
Yes, currently Smart-Seq, Drop-Seq, Fludigm...

Yes, currently Smart-Seq, Drop-Seq, Fludigm single cell RNA-Seq, Illumina Bio-Rad single cell and some other techniques use Nextera to prep libraries from cDNA.
Forum: Sample Prep / Library Generation 10-16-2017, 12:33 AM
Replies: 10
Views: 855
Posted By nucacidhunter
I hope that you have asked all C residues to be...

I hope that you have asked all C residues to be synthesized with mC to prevent C conversion to U during bisulfite treatment (which is very expensive) unless you are using techniques that does not...
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