Forum: Ion Torrent
12-12-2012, 07:33 AM
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Replies: 2
Views: 4,486
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Forum: RNA Sequencing
10-31-2012, 02:47 AM
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Replies: 7
Views: 3,192
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...
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Forum: RNA Sequencing
10-29-2012, 01:51 AM
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Replies: 7
Views: 3,192
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Forum: Illumina/Solexa
04-20-2010, 04:43 AM
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Replies: 1
Views: 1,750
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...
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Forum: 454 Pyrosequencing
02-12-2010, 12:55 AM
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Replies: 6
Views: 9,616
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...
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Forum: 454 Pyrosequencing
02-10-2010, 06:33 AM
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Replies: 6
Views: 9,616
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...
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Forum: 454 Pyrosequencing
01-11-2010, 06:23 AM
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Replies: 8
Views: 4,097
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...
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Forum: 454 Pyrosequencing
01-11-2010, 04:16 AM
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Replies: 8
Views: 4,097
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...
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Forum: 454 Pyrosequencing
01-05-2010, 06:43 AM
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Replies: 15
Views: 9,407
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Forum: Illumina/Solexa
10-27-2009, 03:54 AM
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Replies: 25
Views: 10,782
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...
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Forum: 454 Pyrosequencing
10-27-2009, 12:58 AM
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Replies: 18
Views: 8,237
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...
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Forum: 454 Pyrosequencing
10-26-2009, 04:18 AM
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Replies: 18
Views: 8,237
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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