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Search: Posts Made By: Daytwa
Forum: Literature Watch 02-25-2016, 04:03 PM
Replies: 19
Views: 13,349
Posted By Daytwa
Sorry! It never worked for us.

Sorry! It never worked for us.
Forum: Bioinformatics 04-16-2015, 12:02 PM
Replies: 0
Views: 978
Posted By Daytwa
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...
Forum: RNA Sequencing 02-25-2015, 01:27 PM
Replies: 0
Views: 1,286
Posted By Daytwa
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...
Forum: Bioinformatics 05-20-2014, 07:36 PM
Replies: 0
Views: 885
Posted By Daytwa
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...
Forum: Bioinformatics 04-17-2014, 04:17 PM
Replies: 0
Views: 958
Posted By Daytwa
Why do Cufflinks and Cuffdiff use different default normalization methods?

By default Cufflinks calculates FPKM by using all aligned hits in the denominator while Cuffdiff calculates FPKM by using only reads aligned to a reference transcript.

From reading through other...
Forum: Bioinformatics 03-29-2014, 05:21 PM
Replies: 0
Views: 1,070
Posted By Daytwa
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...
Forum: RNA Sequencing 02-27-2014, 06:37 AM
Replies: 1
Views: 1,208
Posted By Daytwa
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...
Forum: Sample Prep / Library Generation 02-07-2014, 10:58 AM
Replies: 1
Views: 1,575
Posted By Daytwa
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...
Forum: Illumina/Solexa 05-03-2013, 01:40 PM
Replies: 1
Views: 1,202
Posted By Daytwa
Loading more than 10 pmol onto a two lane flowcell

Has anyone tried running more than 10 pmol (the suggested amount) on the two land flowcell?

We ran one run at 10 pmol and hit 250 million paired end reads. Even though that's right in the correct...
Forum: Sample Prep / Library Generation 04-13-2013, 06:00 PM
Replies: 0
Views: 1,641
Posted By Daytwa
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...
Forum: Literature Watch 01-05-2013, 02:29 PM
Replies: 19
Views: 13,349
Posted By Daytwa
Has anyone made any progress with this protocol?...

Has anyone made any progress with this protocol? We can't yield higher than ~10 ng of RNA.
Forum: Sample Prep / Library Generation 01-05-2013, 01:45 PM
Replies: 0
Views: 1,306
Posted By Daytwa
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...
Forum: Sample Prep / Library Generation 05-10-2012, 06:52 AM
Replies: 7
Views: 3,904
Posted By Daytwa
@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...
Forum: Sample Prep / Library Generation 05-10-2012, 06:25 AM
Replies: 7
Views: 3,904
Posted By Daytwa
@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....
Forum: Sample Prep / Library Generation 10-12-2011, 08:39 AM
Replies: 7
Views: 3,904
Posted By Daytwa
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?...
Forum: Illumina/Solexa 01-24-2011, 06:59 AM
Replies: 7
Views: 2,717
Posted By Daytwa
Thanks for the responses so far. Any idea...

Thanks for the responses so far.

Any idea why the Solid and Solexa protocols differ in regards to how they open the circularized product (Restriction enzyme vs shearing)?
Forum: Illumina/Solexa 01-23-2011, 11:46 PM
Replies: 7
Views: 2,717
Posted By Daytwa
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...
Forum: Illumina/Solexa 12-20-2010, 02:57 PM
Replies: 5
Views: 4,831
Posted By Daytwa
@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!
Forum: Illumina/Solexa 12-14-2010, 05:10 PM
Replies: 2
Views: 2,083
Posted By Daytwa
Paired End Protocol for Very Low Abundance Samples

All,

This is my first post after sometime lurking. Utilizing what I've seen in the forum I think I have suitably adapted the standard Illumina ChIP paired end protocol to achieve our goal of...
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