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Forum: Illumina/Solexa 02-15-2018, 11:27 AM
Replies: 6
Views: 505
Posted By pmiguel
Well, one thing. You mentioned you changed the...

Well, one thing. You mentioned you changed the sample sheet after the run. What sample sheet was used by the MiSeq during the run?

The length of the i7 index read is determined by the length of...
Forum: Illumina/Solexa 02-15-2018, 11:22 AM
Replies: 6
Views: 505
Posted By pmiguel
Nothing leaps out at me as being wrong with that...

Nothing leaps out at me as being wrong with that sample sheet. You will probably need to take this up with Illumina.

--
Phillip
Forum: Illumina/Solexa 02-15-2018, 04:43 AM
Replies: 6
Views: 505
Posted By pmiguel
What was the error? -- Phillip

What was the error?

--
Phillip
Forum: Bioinformatics 02-13-2018, 12:10 PM
Replies: 1
Views: 536
Posted By pmiguel
"something" being "command". That is, "command...

"something" being "command". That is, "command not found". Could we see the result of:

ls ../cap3

If that exists, then try:

chmod +x ../cap3

prior to rerunning your command.
--
Forum: Sample Prep / Library Generation 02-13-2018, 09:32 AM
Replies: 68
Views: 38,836
Posted By pmiguel
The NexteraXT index kit could work--however the...

The NexteraXT index kit could work--however the standard ATACseq protocol expects the primer concentrations to be ~5X higher than they are supplied in the NexteraXT index kits. To find this out we...
Forum: Bioinformatics 01-26-2018, 04:11 AM
Replies: 2
Views: 353
Posted By pmiguel
I've never understood why people think that...

I've never understood why people think that pair-merging prior to de novo assembly is a good idea. Seems like were that the case, assembly engines would just do pair-merging themselves prior to other...
Forum: Bioinformatics 01-25-2018, 08:29 AM
Replies: 14
Views: 757
Posted By pmiguel
Those looks like they are contigs created by the...

Those looks like they are contigs created by the program SPADEs. SPADEs is a very good de novo assembler that should have been able to easily assemble an influenza genome.

That said, the extremely...
Forum: Bioinformatics 01-24-2018, 06:48 AM
Replies: 14
Views: 757
Posted By pmiguel
I agree with GenoMax. If you have contig files...

I agree with GenoMax. If you have contig files then it is likely that most of the work is done and all you need to do is take your contig file and blast it against the resource that GenoMax links to....
Forum: Bioinformatics 01-24-2018, 04:21 AM
Replies: 14
Views: 757
Posted By pmiguel
The .fastq data would be the raw reads. "contigs"...

The .fastq data would be the raw reads. "contigs" implies that some program has been used to combine the reads. I would suggest you start with the contig files. They will probably be in a simple...
Forum: Bioinformatics 01-23-2018, 01:47 PM
Replies: 14
Views: 757
Posted By pmiguel
If you got 24 contig files, then aren't the...

If you got 24 contig files, then aren't the sequences of your strains in those? How many contigs did you get per sample?

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Phillip
Forum: Sample Prep / Library Generation 01-18-2018, 08:23 AM
Replies: 14
Views: 4,819
Posted By pmiguel
Ah. So the genomic DNA stays attached to the...

Ah. So the genomic DNA stays attached to the beads, but still works fine for downstream manipulation. Interesting...

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Phillip
Forum: Sample Prep / Library Generation 01-17-2018, 06:25 AM
Replies: 14
Views: 4,819
Posted By pmiguel
Hi melop, What are your elution conditions? ...

Hi melop,
What are your elution conditions?
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Phillip
Forum: Illumina/Solexa 01-10-2018, 08:29 AM
Replies: 1
Views: 376
Posted By pmiguel
Yes, this is possible. Some companies will even...

Yes, this is possible. Some companies will even anneal the adapters for you and ship them in that format.
Usually you would want to put a phosphorothioate linkage for the 3' T overhang base to...
Forum: Illumina/Solexa 01-08-2018, 12:22 PM
Replies: 4
Views: 722
Posted By pmiguel
What sort of adapters are these that you are...

What sort of adapters are these that you are using?
Illumina instruments that prime i5 using the flow cell oligos (including the MiSeq) will always produce i5 sequence from a library molecule. But...
Forum: Bioinformatics 12-21-2017, 07:30 AM
Replies: 3
Views: 933
Posted By pmiguel
You could always hard-clip your reads to 50...

You could always hard-clip your reads to 50 bases.

--
Phillip
Forum: Sample Prep / Library Generation 12-12-2017, 09:23 AM
Replies: 3
Views: 562
Posted By pmiguel
Hi arctan, You might want to take an aliquot of...

Hi arctan,
You might want to take an aliquot of your sample, heat it to 70 oC for a few minutes to denature it's secondary structure, snap cool and then retest for the presence of genomic DNA.
...
Forum: General 11-10-2017, 05:29 AM
Replies: 7
Views: 6,243
Posted By pmiguel
I would speculate as follows: High or low %GC...

I would speculate as follows: High or low %GC will increase the chances of stable single-stranded structures (EG stem loops) forming. In vivo, polymerases will act in concert with a host of other...
Forum: Illumina/Solexa 10-20-2017, 09:39 AM
Replies: 1
Views: 468
Posted By pmiguel
TruSeq® Stranded mRNA LT (Illumina) is designed...

TruSeq® Stranded mRNA LT (Illumina) is designed to fragment long RNAs using heat and divalent cations and then prime 1st and 2nd strand synthesis using hexamer primers.

miRNAs are a specific type...
Forum: Illumina/Solexa 10-18-2017, 08:38 AM
Replies: 10
Views: 1,560
Posted By pmiguel
For Nextera XT, we have always used the Illumina...

For Nextera XT, we have always used the Illumina primers.
Your logic (above) looks sound to me.

--
Phillip
Forum: Sample Prep / Library Generation 10-18-2017, 07:15 AM
Replies: 3
Views: 626
Posted By pmiguel
Thanks for that follow-up. Interesting. -- ...

Thanks for that follow-up. Interesting.

--
Phillip
Forum: Sample Prep / Library Generation 10-17-2017, 09:43 AM
Replies: 3
Views: 626
Posted By pmiguel
Hi patkrat, Yes, the apparently longer products...

Hi patkrat,
Yes, the apparently longer products are likely "bubble products". It is hypothesized that bubble products form during PCR when the concentration of denatured products is high enough to...
Forum: Sample Prep / Library Generation 10-13-2017, 08:48 AM
Replies: 10
Views: 1,036
Posted By pmiguel
Hi Emil, I would strongly recommend that you...

Hi Emil,
I would strongly recommend that you verify this yourself by aligning your p5 and the reverse (in the 3' - 5' direction) of your p7 sequence. You will see the terminal 12 bases on one side...
Forum: Illumina/Solexa 10-13-2017, 08:40 AM
Replies: 26
Views: 2,963
Posted By pmiguel
Well, not as different as you might think. Please...

Well, not as different as you might think. Please see the attachment.

--
Phillip
Forum: Sample Prep / Library Generation 10-11-2017, 02:09 PM
Replies: 10
Views: 1,036
Posted By pmiguel
Hi Emil, Most TruSeq Illumina kits use the...

Hi Emil,
Most TruSeq Illumina kits use the Y-adapters. The common "TruSeq" DNA and RNAseq ones will offer a normal single index kit option (usually going up to 24 indexes) or a "high thoughput" one...
Forum: Sample Prep / Library Generation 10-11-2017, 10:01 AM
Replies: 10
Views: 1,036
Posted By pmiguel
Is this a Y-adapter design? If so, the P7 and P5...

Is this a Y-adapter design? If so, the P7 and P5 need to anneal over the last 12 bases of the P5/first 12 bases of the P7, with a 3' "T" overhang to work with many Illumina work flows. When you add 8...
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