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Forum: Sample Prep / Library Generation 12-07-2011, 12:55 PM
Replies: 18
Views: 26,412
Posted By upenn_ngs
Attached is the most recent working protocol...

Attached is the most recent working protocol including primer sequence and modifications. This protocol is buried in another thread somewhere in the forum. First half = sample prep; second half =...
Forum: Sample Prep / Library Generation 10-27-2011, 06:13 AM
Replies: 22
Views: 11,357
Posted By upenn_ngs
Quick question. How does ribo-zero target rRNA? ...

Quick question. How does ribo-zero target rRNA? I do not want to lose mRNA that is bound in ribosome complex.
Forum: Sample Prep / Library Generation 09-07-2011, 12:30 PM
Replies: 2
Views: 2,002
Posted By upenn_ngs
updated protocol, changes include: *update...

updated protocol, changes include:

*update to pooled enrichment concentration
*fixed single-base typo in P7_barcode_adapter
Forum: Illumina/Solexa 07-29-2011, 09:26 AM
Replies: 0
Views: 1,145
Posted By upenn_ngs
exome enriched mate-pair libraries

We would like to detect large structural variations affecting genes, has anyone had success preparing mate-pair libraries and then enriching for exonic fragments?
Forum: Sample Prep / Library Generation 04-13-2011, 11:47 AM
Replies: 2
Views: 2,002
Posted By upenn_ngs
Agilent Pooled-Enrichment Protocol

The more sequence, the better. See attached.

Have pooled as many as 5 individuals into a single capture, currently waiting for analysis.
Forum: Illumina/Solexa 03-24-2011, 12:53 PM
Replies: 2
Views: 2,076
Posted By upenn_ngs
thanks krobison, but i'm a bit dense. could you...

thanks krobison, but i'm a bit dense. could you spell out your explanations with more detail. personally i am interested in detecting structural variation from exome data.
Forum: Illumina/Solexa 03-24-2011, 07:56 AM
Replies: 2
Views: 2,076
Posted By upenn_ngs
structural variation and read length

What is the current thinking about paired read length (100bp or 150bp) and detection of structural variants? Are the longer 150bp reads advantageous? And how might the library insert size influence...
Forum: Illumina/Solexa 03-21-2011, 08:50 AM
Replies: 45
Views: 25,479
Posted By upenn_ngs
it is also possible to saturate the bioanalyzer...

it is also possible to saturate the bioanalyzer assay. if the library is highly concentrated, then 1ul might introduce too many dna fragments. consequently, these fragments may not travel evenly...
Forum: Sample Prep / Library Generation 03-16-2011, 05:33 AM
Replies: 2
Views: 2,572
Posted By upenn_ngs
so will this will be cDNA resequencing? I am...

so will this will be cDNA resequencing? I am unsure from your description.

you could try scaling down (1/2 or 1/4) reaction volumes for library preparation; however, i don't have personal...
Forum: Genomic Resequencing 03-07-2011, 07:57 AM
Replies: 4
Views: 5,642
Posted By upenn_ngs
could be the probes used to target additional...

could be the probes used to target additional loci in v2 are less stringent, or a difference in sample prep
Forum: Genomic Resequencing 03-07-2011, 05:32 AM
Replies: 4
Views: 5,642
Posted By upenn_ngs
what is the % reads on target including +/-100bp?...

what is the % reads on target including +/-100bp? if this metric shows >90% reads on target then i suspect your coverage issue can be resolved by narrowing the gDNA insert size based on your...
Forum: Sample Prep / Library Generation 03-07-2011, 05:22 AM
Replies: 1
Views: 2,450
Posted By upenn_ngs
the sequencing adapters add ~60bp to either end...

the sequencing adapters add ~60bp to either end of the fragments gDNA (referred to as the "insert"). for 2x100bp sequencing, an insert size of 300bp (+120bp adapters =420bp total) ensures that the...
Forum: Sample Prep / Library Generation 03-04-2011, 05:22 AM
Replies: 2
Views: 3,353
Posted By upenn_ngs
For agilent capture we have pooled using...

For agilent capture we have pooled using equimolar concentrations based on the Bioanalyzer. Read coverage was consistent across indices (within 2%).
Forum: Illumina/Solexa 02-25-2011, 08:58 AM
Replies: 1
Views: 2,098
Posted By upenn_ngs
Making the indexed-adapters on your own can be...

Making the indexed-adapters on your own can be simple and straightforward. See attached.
Forum: Sample Prep / Library Generation 02-24-2011, 12:31 PM
Replies: 18
Views: 26,412
Posted By upenn_ngs
In my hands, I was unable to reliably produce...

In my hands, I was unable to reliably produce libraries with the three primer scheme. But move forward and hold your fingers crossed.
Forum: Illumina/Solexa 02-24-2011, 08:54 AM
Replies: 43
Views: 135,033
Posted By upenn_ngs
To prevent the oligo from serving as a primer for...

To prevent the oligo from serving as a primer for extension during post-hybridization PCR.
Forum: Illumina/Solexa 02-24-2011, 06:26 AM
Replies: 43
Views: 135,033
Posted By upenn_ngs
Short answer: Final [ ] for each oligo 50uM ...

Short answer: Final [ ] for each oligo 50uM

The truseq multiplexed libraries include the index before the enrichment. To maintain the integrity of these barcodes, we use a short-oligo strategy to...
Forum: Sample Prep / Library Generation 02-16-2011, 05:23 AM
Replies: 4
Views: 2,177
Posted By upenn_ngs
Have you considered exome hybridizing with the...

Have you considered exome hybridizing with the circularized mate pair library (before cutting)?
Forum: SOLiD 02-11-2011, 06:37 AM
Replies: 11
Views: 3,079
Posted By upenn_ngs
Right, I suppose the emulsion PCR could be done...

Right, I suppose the emulsion PCR could be done using microbeads with oligos homologous to the illumina adapters.

In addition, the blunt-end ligation scheme of SOLiD prep is not close to the...
Forum: SOLiD 02-10-2011, 10:38 AM
Replies: 11
Views: 3,079
Posted By upenn_ngs
Illumina-compatible runs

Why hasn't ABI produced flow cells compatible with Illumina sample preparations? At our facility the SOLiD machine has yet to be turned on, while the HiSeqs constantly run.

I do not believe...
Forum: Illumina/Solexa 02-03-2011, 05:48 AM
Replies: 45
Views: 25,479
Posted By upenn_ngs
You can certainly collect multiple fractions. We...

You can certainly collect multiple fractions. We did this before finding a run time that gives a reliable size range, though there is still a degree of variability between each sample and each gel. ...
Forum: Illumina/Solexa 02-03-2011, 05:11 AM
Replies: 45
Views: 25,479
Posted By upenn_ngs
We are preparing samples for exome enrichment...

We are preparing samples for exome enrichment followed by 2x100bp reads. We collect TruSeq libraries after 18:10 on the E-gel, and amplification typically yields a mean size ~380bp (+/- 20bp, s.d....
Forum: Illumina/Solexa 01-28-2011, 12:47 PM
Replies: 2
Views: 3,908
Posted By upenn_ngs
I believe the pre-capture PCR primer is...

I believe the pre-capture PCR primer is equivalent to the Illumina Multiplexing PCR Primer 2.0

The post-capture PCR primer is simply the Illumina Multiplexing PCR Primer 1.0 .

I cannot confirm...
Forum: Illumina/Solexa 01-27-2011, 07:58 AM
Replies: 45
Views: 25,479
Posted By upenn_ngs
We have used TruSeq adapters and E-Gel...

We have used TruSeq adapters and E-Gel size-selection, the input gDNA is 1ug (vs. 3ug for the previous protocol), so the gel band will look less intense. Additionally, the y-adapters for TruSeq are...
Forum: Sample Prep / Library Generation 01-25-2011, 05:10 AM
Replies: 23
Views: 13,651
Posted By upenn_ngs
from our experience, a minimum 4-4.5 Gb needs to...

from our experience, a minimum 4-4.5 Gb needs to be collected for the Agilent exome. it does not appear that MiSeq was designed with exome resequencing in mind; but certainly a smaller targeted...
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