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Forum: Illumina/Solexa 06-15-2010, 06:10 PM
Replies: 7
Views: 3,442
Posted By gaster
Thanks for your responses, Link1. Does the...

Thanks for your responses, Link1.

Does the 5' adaptors have to be RNA? It seem cheaper and more stable to use DNA.
Forum: Illumina/Solexa 06-15-2010, 11:33 AM
Replies: 7
Views: 3,442
Posted By gaster
Sequence for directional mRNA seq adaptors?

I am looking for the sequences of the sRNA3', sRNA 5', and SRA Rt primer mentioned in the Illumina Directional mRNA seq adaptor. These sequences are listed in the main "sequence" thread but I...
Forum: Illumina/Solexa 05-12-2010, 11:17 AM
Replies: 16
Views: 11,422
Posted By gaster
me too

I have had the same problem. I am using a no-amp approach and with the two libraries I have tried the ligation efficiency (including recovery efficiency) is less than 1%. I have tried both NEB...
Forum: Illumina/Solexa 04-15-2010, 07:44 AM
Replies: 10
Views: 9,466
Posted By gaster
It is worth pointing out that the Sanger paper...

It is worth pointing out that the Sanger paper listed above does not use the same ss-mRNAseq method used in the Illumina protocol. Also, in the method the Sanger center uses in that paper, there are...
Forum: 454 Pyrosequencing 08-13-2009, 07:23 AM
Replies: 7
Views: 2,826
Posted By gaster
Thanks for your reply. We think these are...

Thanks for your reply. We think these are perfect repeats of 90 basepairs. It is just one region so we would rather not do another 454 or a Solexa run. Any ideas?
Forum: 454 Pyrosequencing 08-12-2009, 01:32 PM
Replies: 7
Views: 2,826
Posted By gaster
Sequencing repeats: how to?

Anyone know of a way to sequence repeats? We have a large 1000bp region of repeats. We can't sequence though it with either 454 or even ABI. Seems like there might some techniques out there for...
Forum: 454 Pyrosequencing 07-28-2009, 05:46 AM
Replies: 5
Views: 3,227
Posted By gaster
Yes, I think you can use the 3'- 5' exo- Klenow...

Yes, I think you can use the 3'- 5' exo- Klenow Fragment. Check out NEB's latest flyer. I am told the Bst polymerase adds dAs.

Good luck!
Forum: Bioinformatics 07-06-2009, 01:33 PM
Replies: 0
Views: 2,326
Posted By gaster
comparing sequences and mapping annotations?

We have two sequences with a little divergence and no large-scale rearrangments (ie gene order and number are probably maintained). One sequence is already annotated. We are trying to find a way to...
Forum: 454 Pyrosequencing 06-24-2009, 05:25 PM
Replies: 5
Views: 3,227
Posted By gaster
Thanks, Marklar. I will check it out.

Thanks, Marklar. I will check it out.
Forum: 454 Pyrosequencing 06-23-2009, 10:32 AM
Replies: 5
Views: 3,227
Posted By gaster
Anyone know what the fill-in enzyme is?

Does anyone know the enzyme Roche uses for the fill-in reaction in the gsflx library prep kit? Anyone know an enzyme that does the same thing?

This is to fill-in a phosphodiester bond on one...
Forum: 454 Pyrosequencing 05-28-2009, 07:21 AM
Replies: 6
Views: 4,468
Posted By gaster
Thanks, Sklages. The metrics files don't seem to...

Thanks, Sklages. The metrics files don't seem to have info about coverage, right?

The 454AlignmentInfo.tsz files only gives me partial info on one contig. Is that right? It can't be. Any...
Forum: 454 Pyrosequencing 05-28-2009, 07:17 AM
Replies: 6
Views: 4,468
Posted By gaster
Thanks for your reply, Flxlex. Couple questions:...

Thanks for your reply, Flxlex. Couple questions: How do you set the g-flag? Is there no way to get min/max coverage?

Thanks.
Forum: 454 Pyrosequencing 05-22-2009, 12:29 PM
Replies: 6
Views: 4,468
Posted By gaster
Summary Statistics in Newbler?

The Mapper output comes with handy xls file with a lot of great statistics in it. The Newbler output doesn't seem to have anything like this? I am particularly looking for statistics about the...
Forum: 454 Pyrosequencing 05-22-2009, 11:55 AM
Replies: 3
Views: 42,681
Posted By gaster
I don't quite understand. I haven't done a...

I don't quite understand. I haven't done a Titanium prep yet, but previously you just wanted the non-biotinylated ssDNA. The biotin-bound strand was discarded. This step makes sure the adaptors...
Forum: 454 Pyrosequencing 05-22-2009, 07:01 AM
Replies: 3
Views: 42,681
Posted By gaster
I'm not sure you will be able to do that with...

I'm not sure you will be able to do that with quantities that small. I would suggest you use genomiphi or something to amplify your DNA.


If you want to give it try, you should find some other...
Forum: 454 Pyrosequencing 05-12-2009, 06:13 AM
Replies: 1
Views: 1,790
Posted By gaster
This sounds like it has taking too long. I don't...

This sounds like it has taking too long. I don't know about your specific assembly, but my experience running Mapper off our server and assembling small <1mb genomes is that is very fast-- less than...
Forum: 454 Pyrosequencing 05-11-2009, 12:43 PM
Replies: 0
Views: 1,906
Posted By gaster
GS Mapper output to consed shows dimmed ends with good sequence?

Somewhat serendipitously I was able to sequence a genome to 60X coverage. I used mapper to align it to a good reference sequence. The results were 6 contigs. When I opened the ace file in the...
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