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Forum: Sample Prep / Library Generation 06-28-2017, 10:37 AM
Replies: 12
Views: 10,599
Posted By kerplunk412
The beads should be fine.

The beads should be fine.
Forum: Bioinformatics 11-17-2016, 10:43 AM
Replies: 5
Views: 1,436
Posted By kerplunk412
This. Also, the small RNA adapter is different...

This. Also, the small RNA adapter is different from the "standard" Illumina adapter, so make sure you trimmed the correct adapter sequence.
Forum: Sample Prep / Library Generation 11-16-2016, 12:00 PM
Replies: 4
Views: 1,623
Posted By kerplunk412
I have Covaris digested DNA of 1-2 kb and it...

I have Covaris digested DNA of 1-2 kb and it works well. I think your problem is #2.
Forum: Sample Prep / Library Generation 10-27-2016, 06:51 AM
Replies: 5
Views: 2,147
Posted By kerplunk412
In my experience the yield from the E-gel is...

In my experience the yield from the E-gel is terrible, which makes sense as it doesn't take DNA very long to cross the buffer well. The Pippin should be able to give you as good or better resolution...
Forum: Sample Prep / Library Generation 10-27-2016, 06:34 AM
Replies: 8
Views: 2,820
Posted By kerplunk412
The Corning Spin-X tubes work well but are not...

The Corning Spin-X tubes work well but are not cheap. Of course you could always avoid the tedious gel step completely with the NEXTflex Small RNA Seq Kit v3...
Forum: Sample Prep / Library Generation 09-21-2016, 07:33 AM
Replies: 9
Views: 1,973
Posted By kerplunk412
Unless there is a large amount of excess PCR...

Unless there is a large amount of excess PCR primer compared to actual library product it should not occupy enough primers on the flow cell to make any difference. The flow cell has enough primers on...
Forum: Sample Prep / Library Generation 09-20-2016, 11:00 AM
Replies: 9
Views: 1,973
Posted By kerplunk412
TriLink has indeed modified the adapters to...

TriLink has indeed modified the adapters to greatly reduce adapter-dimer formation, but I am not sure exactly how it works. In my experience their kit works well but still has big issues with bias.
...
Forum: Illumina/Solexa 08-19-2016, 12:21 PM
Replies: 10
Views: 1,630
Posted By kerplunk412
Can't you just extend the sequencing primers in...

Can't you just extend the sequencing primers in the other direction, into the P5/P7? Another option would be to add an LNA base or two, but that would be more expensive.
Forum: Sample Prep / Library Generation 08-19-2016, 12:02 PM
Replies: 2
Views: 723
Posted By kerplunk412
Hi Sunitha, I do not know the concentration...

Hi Sunitha,
I do not know the concentration of the primers in the Illumina kit, but you can probably estimate it fairly accurately using a Nanodrop or Qubit ssDNA assay.
FYI, you can get...
Forum: Sample Prep / Library Generation 08-19-2016, 11:48 AM
Replies: 4
Views: 1,390
Posted By kerplunk412
To expand on this a little, it appears that all...

To expand on this a little, it appears that all Bioanalyzer DNA and RNA chips are the same chip, just with a different sticker.
Forum: RNA Sequencing 08-10-2016, 11:26 AM
Replies: 3
Views: 2,164
Posted By kerplunk412
I think this is what you need: cutadapt -a...

I think this is what you need:

cutadapt -a AGATCGGAAGAG -o YOUR_FILE.trim1.fq --minimum-length 15 YOUR_FILE.fastq.gz

You don't need to put in the index sequence, as cutadapt will remove...
Forum: Sample Prep / Library Generation 07-18-2016, 11:32 AM
Replies: 4
Views: 1,358
Posted By kerplunk412
I know that to select small RNA libraries of ~150...

I know that to select small RNA libraries of ~150 bp and exclude adapter-dimer at ~130 bp you must use settings way different from expected (115 bp-170 bp collection range), so I am not surprised...
Forum: Bioinformatics 07-08-2016, 07:21 AM
Replies: 6
Views: 1,800
Posted By kerplunk412
Yes, with most RNA-Seq protocols the small RNAs...

Yes, with most RNA-Seq protocols the small RNAs are eliminated in the cleanup steps. Also, the typical first and second strand synthesis strategies will rarely capture the entirety of an RNA...
Forum: Illumina/Solexa 06-21-2016, 12:49 PM
Replies: 26
Views: 5,799
Posted By kerplunk412
The 600 cycle v3 kits were showing really poor...

The 600 cycle v3 kits were showing really poor base qualities toward the end of reads. I am not sure if Illumina has resolved this yet.
Forum: Sample Prep / Library Generation 06-17-2016, 09:26 AM
Replies: 9
Views: 1,973
Posted By kerplunk412
Response sent. Also, to be fair I should...

Response sent.

Also, to be fair I should mention some of the other kits out there and the pros and cons.

NEB: Cost effective, lower bias than TruSeq, but substantially more bias than NEXTflex...
Forum: Sample Prep / Library Generation 06-15-2016, 07:49 AM
Replies: 9
Views: 1,973
Posted By kerplunk412
The RNA looks good to me, but the libraries seem...

The RNA looks good to me, but the libraries seem to be just adapter-dimer. How much RNA input did you use?

Also, I would like to mention that the TruSeq kit is about the worst Small RNA-Seq kit...
Forum: Core Facilities 03-30-2016, 01:25 PM
Replies: 10
Views: 6,881
Posted By kerplunk412
I contacted a collaborator today who has been...

I contacted a collaborator today who has been using Bioo Scientific small RNA kits and just had samples sequenced on a HiSeq 4000, and although they haven't analyzed the data in detail yet everything...
Forum: Sample Prep / Library Generation 02-08-2016, 11:18 AM
Replies: 9
Views: 2,464
Posted By kerplunk412
gDNA contamination isn't much of a problem for...

gDNA contamination isn't much of a problem for RNA-Seq, as the contaminating gDNA is too large to be well represented in the final library (or to cluster efficiently on the flow cell if it does make...
Forum: Sample Prep / Library Generation 01-27-2016, 02:29 PM
Replies: 4
Views: 1,894
Posted By kerplunk412
Happy to help, and thanks for posting your...

Happy to help, and thanks for posting your results and protocol.
Forum: Sample Prep / Library Generation 01-25-2016, 02:54 PM
Replies: 4
Views: 1,894
Posted By kerplunk412
Hi Mat. Welcome the the forum, and you didn't do...

Hi Mat. Welcome the the forum, and you didn't do anything wrong on the post, it is very clear. I have never used this kit, but some things you may want to consider are using less enzyme or shorter...
Forum: Sample Prep / Library Generation 01-25-2016, 02:41 PM
Replies: 3
Views: 1,927
Posted By kerplunk412
I say re-run it and see what happens. With...

I say re-run it and see what happens. With something this strange I always like to make sure the result is repeatable before looking too much into it.

Also, if you still have the rest of the 1:20...
Forum: Sample Prep / Library Generation 01-12-2016, 03:08 PM
Replies: 5
Views: 1,546
Posted By kerplunk412
The NEXTflex Poly(A) Beads...

The NEXTflex Poly(A) Beads (http://www.biooscientific.com/Next-Gen-Sequencing/Illumina-RNA-Seq/NEXTflex-Poly-A-Beads) from Bioo Scientific work quite well and are very cost-effective. As a...
Forum: Sample Prep / Library Generation 01-12-2016, 03:02 PM
Replies: 9
Views: 2,464
Posted By kerplunk412
I agree that the hump is likely degraded rRNA....

I agree that the hump is likely degraded rRNA. This is often accompanied by a "peak" of degraded products around 100 nt, but I suspect you aren't seeing that here due to your extraction method. This...
Forum: Sample Prep / Library Generation 12-11-2015, 03:45 PM
Replies: 2
Views: 2,774
Posted By kerplunk412
The peak you see at 270 is almost certainly...

The peak you see at 270 is almost certainly phenol contamination, and means any Nanodrop quantification can't be trusted. The good news is that it might not actually be that bad. This amount of...
Forum: Sample Prep / Library Generation 12-02-2015, 03:13 PM
Replies: 8
Views: 3,662
Posted By kerplunk412
In my experience degraded RNA shows a strong peak...

In my experience degraded RNA shows a strong peak around 100 nt on the Bioanalyzer, so that doesn't exactly look like degraded RNA to me. Since your purification removed the <200nt RNAs, the ~100nt...
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