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Forum: Bioinformatics 02-04-2020, 08:00 AM
Replies: 2
Views: 702
Posted By Mike2188
Yes, coverage is extremely high, had several...

Yes, coverage is extremely high, had several million reads from a miseq run covering a small viral genome of ~10kb.

Oh, okay. If it takes the overall highest quality read, and doesn't consider...
Forum: Bioinformatics 02-03-2020, 10:18 AM
Replies: 2
Views: 702
Posted By Mike2188
Read deduplication with extremely high coverage and variant calling

Hello,

I'm currently in a class, and the professor is having us run through some NGS practice data from HIV. I tried to find an exact answer in the forums but couldn't. I feel like the professor...
Forum: Bioinformatics 01-26-2015, 10:26 AM
Replies: 0
Views: 865
Posted By Mike2188
Cufflinks, cuffmerge and strandedness

I have a question, I am currently using cufflinks for multiple bam to generate .gtfs and cuffmerge on these to improve an already existing annotation (Brassica napus). I find, in several hundred...
Forum: Sample Prep / Library Generation 11-17-2014, 07:42 AM
Replies: 3
Views: 3,332
Posted By Mike2188
Those are peaks from chloroplast rRNA, which is...

Those are peaks from chloroplast rRNA, which is common in green tissues -- especially leaf.
Forum: Sample Prep / Library Generation 10-27-2014, 08:41 AM
Replies: 7
Views: 1,775
Posted By Mike2188
Nope, these samples are simple extracted using...

Nope, these samples are simple extracted using Trizol. Old school. I am going to try a kit to see if it changes the results later this week.
Forum: Sample Prep / Library Generation 10-27-2014, 06:26 AM
Replies: 7
Views: 1,775
Posted By Mike2188
In regards to my above comment, here is what...

In regards to my above comment, here is what electropherograms will typically look like in previous instances where I have had severely degraded RNA. However, these samples were heat and chemically...
Forum: Sample Prep / Library Generation 10-27-2014, 06:20 AM
Replies: 7
Views: 1,775
Posted By Mike2188
Thanks, I have worked with low RNA quality...

Thanks,

I have worked with low RNA quality samples before, with RINs ranging from 1-9 on the bioanalyzer, and I have never seen just one small and sharp peak like this before. I would expect RNA...
Forum: Sample Prep / Library Generation 10-26-2014, 09:38 PM
Replies: 7
Views: 1,775
Posted By Mike2188
Thanks for your interest, Here are the full...

Thanks for your interest,

Here are the full reports. You'll find the ladder and everything else looks normal. Some samples were run multiple times, over both chips, and look the same. Some samples...
Forum: Sample Prep / Library Generation 10-26-2014, 05:34 PM
Replies: 7
Views: 1,775
Posted By Mike2188
Weird RNA: Is this degraded or not?

I have been asked to prepare some sequencing libraries for a collaborator from pellets of psuedomonas bacteria that were in stationary phase. I isolated RNA and ran it on the agilent 2100 Bioanalyzer...
Forum: Sample Prep / Library Generation 08-28-2014, 06:19 PM
Replies: 3
Views: 1,438
Posted By Mike2188
Thank you for the replies. They are much...

Thank you for the replies. They are much appreciated. Those kits are quite affordable for poly(A) isolation, but I am working with samples in about the 100-200 ng range, which is below the input for...
Forum: Sample Prep / Library Generation 08-26-2014, 07:11 AM
Replies: 3
Views: 1,438
Posted By Mike2188
Isolation of mRNA

Hi everyone, just a quick question.

I am using a library protocol that uses a magnetic poly(A) isolation of mRNA, followed by a random hexamer based first strand cDNA synthesis step. The poly(A)...
Forum: Sample Prep / Library Generation 08-01-2014, 07:51 AM
Replies: 5
Views: 1,987
Posted By Mike2188
Also, Sara is mistaken, your concentrations are...

Also, Sara is mistaken, your concentrations are sufficient, as they reach or exceed the marker.
Forum: Sample Prep / Library Generation 08-01-2014, 07:46 AM
Replies: 5
Views: 1,987
Posted By Mike2188
A few questions: a) what samples are you...

A few questions:

a) what samples are you working with?
- certain tissues have different forms of contamination, for instance I work with leaf and seed so often carbohydrates may be an issue.
...
Forum: RNA Sequencing 07-31-2014, 08:31 AM
Replies: 3
Views: 1,540
Posted By Mike2188
Starting files: gq2-sequence.fa ...

Starting files:
gq2-sequence.fa
transcripts.fa

Build Genome Index:
bowtie2-build gq2-sequence.fa gq2

Align using tophat and built index:
tophat2 --b2-very-senstive -p 8 gq2 transcripts.fa
Forum: Bioinformatics 07-31-2014, 07:09 AM
Replies: 2
Views: 4,920
Posted By Mike2188
Both cufflinks and cuffdiff will generate FPKM...

Both cufflinks and cuffdiff will generate FPKM data. Cuffdiff will also spit out differentially expressed genes between your samples and take a bit longer.
Forum: RNA Sequencing 07-31-2014, 07:05 AM
Replies: 3
Views: 1,540
Posted By Mike2188
This may be stupid, even if it is one gene, if...

This may be stupid, even if it is one gene, if you are aligning a transcript sequence, shouldn't you use tophat?

I couldn't see an error in what you did.
Forum: Bioinformatics 07-30-2014, 03:48 PM
Replies: 0
Views: 1,203
Posted By Mike2188
Paired end alignments -- What to throw away and what to keep?

I am working with a fairly dense dataset containing 12 samples each containing 45 million paired end reads.

I trimmed and quality filtered these reads using trimmomatic. I obtained two pair end...
Forum: Bioinformatics 07-30-2014, 03:30 PM
Replies: 4
Views: 2,440
Posted By Mike2188
Generally when you are aligning it is best to...

Generally when you are aligning it is best to align to the genome for several reasons:
a) do not bias towards transcriptome or sequences you are aligning too
b) allows for discovery of novel...
Forum: Bioinformatics 07-30-2014, 03:04 PM
Replies: 3
Views: 1,762
Posted By Mike2188
sorry, I put the same link twice... Here is the...

sorry, I put the same link twice... Here is the second link.

http://seqanswers.com/forums/showthread.php?t=24205&page=2

Also, someone mentioned that increasing their RAM helped. Are you working...
Forum: Bioinformatics 07-30-2014, 03:02 PM
Replies: 3
Views: 1,762
Posted By Mike2188
This has been an issue in the past: ...

This has been an issue in the past:

https://groups.google.com/forum/#!topic/tuxedo-tools-users/a3TXz72Z3F8

https://groups.google.com/forum/#!topic/tuxedo-tools-users/a3TXz72Z3F8

I believe it...
Forum: RNA Sequencing 07-30-2014, 02:09 PM
Replies: 5
Views: 5,469
Posted By Mike2188
If you do each file individually then you run...

If you do each file individually then you run into errors during alignments. For instance if I had 100,000 paired end reads in two files forward.fq and reverse.fq and I performed some trimming and...
Forum: Bioinformatics 05-21-2014, 07:00 PM
Replies: 3
Views: 2,283
Posted By Mike2188
I have several laser microdissected subtissues of...

I have several laser microdissected subtissues of a specialized plant structure called the funiculus that connects to a developing seed. I am working with canola. I am looking for differential...
Forum: Bioinformatics 05-21-2014, 03:59 PM
Replies: 3
Views: 2,283
Posted By Mike2188
Transcriptome vs. Genome alignments

I have some RNA-seq data back and I am performing alignments with Bowtie2. The organism I am working with has an available transcriptome assembly as well as genome assembly. I am not interested in...
Forum: General 04-18-2014, 01:24 PM
Replies: 1
Views: 2,238
Posted By Mike2188
Conceptual question of validity of transcriptional profiling

I have a question regarding data handling of RNA-seq experiments and even microarray I guess.

How do these methods take total total cellular expression into account?

For instance say I was...
Forum: Sample Prep / Library Generation 01-23-2014, 09:11 AM
Replies: 5
Views: 4,278
Posted By Mike2188
Some people have sequenced overamplified...

Some people have sequenced overamplified libraries with success if redoing is expensive or not much of an option. Also, people have reannealed their libraries. What happens is the DNA forms ds...
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