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Forum: Ion Torrent 12-12-2012, 07:33 AM
Replies: 2
Views: 4,486
Posted By sulfobus
Is the 200bp read length kit available for the...

Is the 200bp read length kit available for the Proton?
Forum: RNA Sequencing 10-31-2012, 02:47 AM
Replies: 7
Views: 3,192
Posted By sulfobus
We've had chimeric sequences but they were in...

We've had chimeric sequences but they were in distinct groups with the same lengths. From your distribution it looks like most are spread out, although there are two peaks. In our case it was a PCR...
Forum: RNA Sequencing 10-29-2012, 01:51 AM
Replies: 7
Views: 3,192
Posted By sulfobus
Is the read length disitribution for the chimeras...

Is the read length disitribution for the chimeras smooth, or does it have many spikes?
Forum: Illumina/Solexa 04-20-2010, 04:43 AM
Replies: 1
Views: 1,750
Posted By sulfobus
It could be heteroduplexes forming during late...

It could be heteroduplexes forming during late stage PCR or during purification (esp. using chaotropic detergents s.a. guanidine salts in combination with heat). They migrate slower on gel, check...
Forum: 454 Pyrosequencing 02-12-2010, 12:55 AM
Replies: 6
Views: 9,616
Posted By sulfobus
Actually, we don't sequence ourselves but our...

Actually, we don't sequence ourselves but our sequence provider didn't know what could cause this bias. I will bring up the suggestion about software bug with them, and ask them to use the latest...
Forum: 454 Pyrosequencing 02-10-2010, 06:33 AM
Replies: 6
Views: 9,616
Posted By sulfobus
Homopolymer run errors, polyA bias

Pyrosequencing has problems with determining the correct number of bases in a homopolymer stretch. However, we notice that this problem is much more frequent with polyA than polyT/C/G. How can that...
Forum: 454 Pyrosequencing 01-11-2010, 06:23 AM
Replies: 8
Views: 4,097
Posted By sulfobus
Indeed, but it saves some money in reagents and...

Indeed, but it saves some money in reagents and plates. The samples we sequence sometimes only contain ~1000 unique sequences, so even a 16th plate would be oversampling. That's why we use ID-tags...
Forum: 454 Pyrosequencing 01-11-2010, 04:16 AM
Replies: 8
Views: 4,097
Posted By sulfobus
Hi We regularly use up to 10 "in-house MIDs"...

Hi
We regularly use up to 10 "in-house MIDs" for 454, and yes, balancing the load is very difficult. We regularly see 4-fold differences, often more. More accurate quantification might help, but I...
Forum: 454 Pyrosequencing 01-05-2010, 06:43 AM
Replies: 15
Views: 9,407
Posted By sulfobus
Is this kit for amplicon sequencing with titanium...

Is this kit for amplicon sequencing with titanium released yet?
Forum: Illumina/Solexa 10-27-2009, 03:54 AM
Replies: 25
Views: 10,782
Posted By sulfobus
2nd larger DNA band after enrichment PCR

I frequently see this phenomenon, not only realted to Illumina sequencing. I believe it is mostly heteroduplexes, i.e. partially ssDNA that partially hybridize to other strands with a segment of...
Forum: 454 Pyrosequencing 10-27-2009, 12:58 AM
Replies: 18
Views: 8,237
Posted By sulfobus
Titanium - high fraction of short reads

Our truncated sequences starts off correctly, but then suddenly halts. It is no chimera with insertions of primers or adaptors, just truncated correct sequences. We elongate our DNA with the...
Forum: 454 Pyrosequencing 10-26-2009, 04:18 AM
Replies: 18
Views: 8,237
Posted By sulfobus
Question Titanium - high fraction of short reads

Hi

I often see the same phenomenon in my Ti454 sequences: a peak of truncated reads ~50 bp and a lot of short reads <150 bp. It does not appear to be due to some obvious PCR artefact, s.a. primer...
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