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Forum: General 01-23-2018, 11:16 AM
Replies: 0
Views: 1,421
Posted By SDK
Automating consensus sequence generation...?

I have a rather straightforward problem. I'm doing a pop. gen. study which has ~1200 individuals. Using our Miseq, I've generated short (~150b) fragments for these individuals at 5 loci (one...
Forum: Bioinformatics 01-19-2018, 09:13 AM
Replies: 0
Views: 1,192
Posted By SDK
Program to make consensus and call SNP?

Hello All,

I've got 2 distinct but related problems...well 3 problems if you count that I'm not a bioinformatician! I've just completed a pop. gen. microsatellite study using the MiSeq. I used a...
Forum: Sample Prep / Library Generation 07-15-2015, 11:50 AM
Replies: 1
Views: 1,755
Posted By SDK
Squeezing the most out of Nextera XT...?

Hi All,

I'm wondering if anyone has experimented with pushing the limits of Nextera XT in order to get more samples out of a kit? I want to sequence a bunch (~150) of mitogenomes generated via...
Forum: Sample Prep / Library Generation 03-03-2015, 07:58 AM
Replies: 6
Views: 4,034
Posted By SDK
Thanks for the reply SNPsaurus. So, how would you...

Thanks for the reply SNPsaurus. So, how would you choose to fragment? I've never used restriction enzymes before but I assume that is the idea here? Would you choose a common cutter? Thanks for any...
Forum: Sample Prep / Library Generation 02-27-2015, 08:05 AM
Replies: 6
Views: 4,034
Posted By SDK
Sequencing Long-Range Amplicons affordably?

Hi All,

I have a question regarding sequencing long-range amplicons affordably, as the title suggests! So, in short I have about 40 long range PCR amplicons from 40 different species (amplicons...
Forum: Bioinformatics 06-10-2014, 03:35 AM
Replies: 1
Views: 1,319
Posted By SDK
Anyone know a program to detect duplicated regions?

Hi All,

I'm working with on generating denovo mtDNA genomes for many species of an invertebrate taxa of the same genus. These critters have a duplicated non-coding region (~800bp) separated by...
Forum: Bioinformatics 04-14-2014, 08:32 AM
Replies: 6
Views: 2,657
Posted By SDK
Thanks Adrian, I'll give that a try. Have you...

Thanks Adrian, I'll give that a try. Have you ever tried this before? Or heard of it begin done? Would love to hear some 'proof of concept' testimonial!
Cheers,
Forum: Bioinformatics 04-13-2014, 04:28 PM
Replies: 6
Views: 2,657
Posted By SDK
Thanks Adrian. The mtDNA genomes are between...

Thanks Adrian. The mtDNA genomes are between 15-18 kb, however we had multiple PCR's of a smaller size (~5-10 kb) to generate a full genome, then we pooled it together. Basically, my pooled library...
Forum: Bioinformatics 04-13-2014, 10:12 AM
Replies: 6
Views: 2,657
Posted By SDK
Demultiplexing a MiSeq library based on Amplicon Sequence divergence?

Hi All,

My lab works on a lot of diverse taxa. We want to sequence the mtDNA genomes of each using our MiSeq. We generated long-range PCR amplicons for each species, then pooled 10 taxonomically...
Forum: General 02-17-2014, 11:05 AM
Replies: 13
Views: 6,047
Posted By SDK
Phillip, Thanks very much for the reply. We...

Phillip,

Thanks very much for the reply. We have actually done that. We used the sequence generated to make denovo genomes which we used to develop primers for pop. gen. work. Now we want to...
Forum: Illumina/Solexa 02-14-2014, 04:43 AM
Replies: 3
Views: 3,481
Posted By SDK
Thanks guys. I thought beads would be the best...

Thanks guys. I thought beads would be the best choice but feared they may not work on really big amplicons. Great to know you guys have used them with success. Cheers!
Forum: General 02-13-2014, 04:19 PM
Replies: 13
Views: 6,047
Posted By SDK
Saimonara, thanks for the advice, I'll give the...

Saimonara, thanks for the advice, I'll give the "hot start" a try. Youm Ug, I think you're just having fun at my expense. Don't need any of your ridiculous questions or crazy answers, go troll...
Forum: General 02-13-2014, 03:07 PM
Replies: 13
Views: 6,047
Posted By SDK
Thank you Siamonara for your reply. Youm Ug, in...

Thank you Siamonara for your reply. Youm Ug, in the images you see I've loaded 5 ul PCR product. I don't recall what type of pipette tips we use, VWR perhaps.
Forum: General 02-13-2014, 12:00 PM
Replies: 13
Views: 6,047
Posted By SDK
Hi Youm Ug, Thanks for the response: ...

Hi Youm Ug,

Thanks for the response:

1) primers were made by IDT
2) the dNTP actually came with the taq so I assume it should be optimized for it.
3) the taxa is a platyhelminth (flat worm)...
Forum: General 02-13-2014, 08:49 AM
Replies: 13
Views: 6,047
Posted By SDK
Long Range PCR problem...

Hi Guys,

So I'm trying to amplify whole mtDNA of a few species using long-range PCR. I've found a conserved region and designed long (30mer) primers at 3 different sites which are about 120 bp and...
Forum: Illumina/Solexa 02-13-2014, 07:11 AM
Replies: 3
Views: 3,481
Posted By SDK
Cleaning amplicons before Nextera + Miseq run??

Hi Guys,

Quick question: I'm planning on sequencing large amplicons (5-15 kbp) on our MiSeq using the Nextera kit. Wondering the best way to clean the PCR product before I run it through Nextera?...
Forum: Bioinformatics 01-20-2014, 03:48 PM
Replies: 1
Views: 1,233
Posted By SDK
Program to Randomly Chop Sequence files?

Hey Guys,

First post! Anyone know of a program one could use to chop up a sequence file (15kbp; mtDNA genome) into random smaller fragments (~150bp)? I want to chop up the sequences of 5 or 10...
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