Forum: Literature Watch
02-25-2016, 04:03 PM
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Replies: 19
Views: 17,580
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Forum: Bioinformatics
04-16-2015, 12:02 PM
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Replies: 0
Views: 1,288
Negative P-values from MACS2
I am using MACS2 to call open chromatin regions in a MNase-seq dataset. However I'm generating negative p-values after calling broad peaks using the following commands:
macs2 callpeak -t...
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Forum: RNA Sequencing
02-25-2015, 01:27 PM
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Replies: 0
Views: 1,700
Problem with GFOLD
I am using GFOLD to select candidate enriched transcripts from an RNA-seq experiment. Treatment #1 contains two replicates while treatment #2 is a singleton. This produced some unexpected results in...
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Forum: Bioinformatics
05-20-2014, 07:36 PM
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Replies: 0
Views: 1,166
coveragebed problem
I am having trouble with the histogram option in coveragebed. I want to compare coverage of my uniquely aligned RNA-seq reads (list A) to a bed file of repeats (list B). When I run this comparison...
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Forum: Bioinformatics
04-17-2014, 04:17 PM
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Replies: 0
Views: 1,229
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Forum: Bioinformatics
03-29-2014, 05:21 PM
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Replies: 0
Views: 1,379
Tophat2 Mapping Error with transgenic genome
I am trying to align RNA-seq reads to a 'custom' mouse genome which contains an expressed human locus. To do this I concatenated the iGenome FASTA files for the mouse genome with the FASTA sequence...
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Forum: RNA Sequencing
02-27-2014, 06:37 AM
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Replies: 1
Views: 1,636
Tuxedo Pipeline with single condition
I've been asked to run a deep coverage mouse RNA-seq sample through the Tuxedo pipeline as this analysis tool has been used before in the lab. However, we are only looking to determine expression...
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Forum: Sample Prep / Library Generation
02-07-2014, 10:58 AM
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Replies: 1
Views: 1,880
Innaccurate Kapa Quant Standards
On my campus two labs, both with very extensive experience in NGS, have reported that recently purchased Kapa qPCR standards have under-estimated library concentrations. Both sets of libraries were...
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Forum: Illumina/Solexa
05-03-2013, 01:40 PM
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Replies: 1
Views: 1,436
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Forum: Sample Prep / Library Generation
04-13-2013, 06:00 PM
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Replies: 0
Views: 1,973
Unwanted Small (~80bp) Bioanalyzer Peaks
After PCR enriching some libraries I noticed small peaks of 80 bp in all of my samples (see image). I am using NEB's ChIP-seq kit for Illumina. My inserts are ~65 bp. I see this in my negative...
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Forum: Literature Watch
01-05-2013, 02:29 PM
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Replies: 19
Views: 17,580
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Forum: Sample Prep / Library Generation
01-05-2013, 01:45 PM
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Replies: 0
Views: 1,607
LinDA Troubleshooting
Has anyone had consistent success using Shankaranarayanan et al.'s Single-tube linear DNA amplification (LinDA) for robust ChIP-seq protocol?
We have yet to yield more than 10 ng of RNA. This...
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Forum: Sample Prep / Library Generation
05-10-2012, 06:52 AM
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Replies: 7
Views: 4,683
@indu,
I am somewhat confused. You are...
@indu,
I am somewhat confused. You are worried about the concentration of you library vs the input? If that is the case don't be. It is normal for the prep to be somewhat lossy. Yields are never...
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Forum: Sample Prep / Library Generation
05-10-2012, 06:25 AM
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Replies: 7
Views: 4,683
@indu
what i've found works best (especially...
@indu
what i've found works best (especially when constructing low input libraries (~10 ng or less) is two use the ampure xp beads following ligation (i do this 3 times). i then run a ~3% gel....
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Forum: Sample Prep / Library Generation
10-12-2011, 08:39 AM
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Replies: 7
Views: 4,683
Post PCR Enrichment Gel Selection for ChIP-Seq
Hello,
After I PCR enrich ChIP-seq libraries following the Illumina protocol I notice bioanalyzer peaks corresponding to what I think is primer or primer (<100bp). Has anyone else seen this?...
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Forum: Illumina/Solexa
01-24-2011, 06:59 AM
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Replies: 7
Views: 3,125
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Forum: Illumina/Solexa
01-23-2011, 11:46 PM
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Replies: 7
Views: 3,125
Very Large Mate Pair Libraries
I am unclear on why the mate pair library protocol requires gap sizes to be under 5 kb. Is this just due to the limits of column purification or reliable fragmentation?
Has anyone ever tried to...
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Forum: Illumina/Solexa
12-20-2010, 02:57 PM
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Replies: 5
Views: 5,490
@csquared
If possible can you please...
@csquared
If possible can you please elaborate on this final point. What would be a good yardstick by which to judge whether a library is pristine? Thanks for any advice you may have!
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Forum: Illumina/Solexa
12-14-2010, 05:10 PM
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Replies: 2
Views: 2,562
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