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Forum: Sample Prep / Library Generation 11-27-2012, 11:45 AM
Replies: 7
Views: 1,481
Posted By mnkyboy
I have had good success using v-bottom plates...

I have had good success using v-bottom plates with the AMPure and the Ambion magnet. That magnet is compatible with PCR plates too if you prefer those.

I prefer the Sarstedt v-well bottom plates.
Forum: General 10-31-2012, 11:54 AM
Replies: 2
Views: 1,676
Posted By mnkyboy
I think you meant this: ...

I think you meant this:

http://lmgtfy.com/?q=bedtools
Forum: Bioinformatics 10-19-2012, 01:49 PM
Replies: 5
Views: 15,495
Posted By mnkyboy
It is just a text file so save it as .fa or...

It is just a text file so save it as .fa or .fasta

If you need a fasta per sequence you will have to write a script, or use some sort of replace function. If you just want the whole file a fasta...
Forum: Bioinformatics 10-19-2012, 01:35 PM
Replies: 5
Views: 15,495
Posted By mnkyboy
All you need to make a FASTA is to make a > on...

All you need to make a FASTA is to make a > on the first line.

http://en.wikipedia.org/wiki/FASTA_format

You cannot got from FASTA to FASTQ.
Forum: Bioinformatics 10-16-2012, 01:59 PM
Replies: 13
Views: 2,297
Posted By mnkyboy
The answer is D.

The answer is D.
Forum: General 10-05-2012, 10:16 AM
Replies: 5
Views: 3,126
Posted By mnkyboy
We get reproducibility from replicates, so we...

We get reproducibility from replicates, so we measure that as success.

Also publications. As for representativity do you mean numbers of small RNAs (diversity)?

Our systems are dominated by a...
Forum: General 10-05-2012, 10:09 AM
Replies: 5
Views: 3,126
Posted By mnkyboy
I have had success with both mirVana and Trizol...

I have had success with both mirVana and Trizol for mamallian cells and tissues.

Since we want do do whole transcriptome and small, we have just switched to the normal Trizol protocol because it...
Forum: Ion Torrent 10-04-2012, 01:26 PM
Replies: 7
Views: 7,814
Posted By mnkyboy
It is not in the documentation, which is why it...

It is not in the documentation, which is why it is confusing. I think the default is -Q 64 and if you use the other you have to do -Q 33. I do not think it supports anything else.
Forum: Ion Torrent 10-04-2012, 12:52 PM
Replies: 7
Views: 7,814
Posted By mnkyboy
I think it is -Q 33 or -Q 64 that specifies which...

I think it is -Q 33 or -Q 64 that specifies which PHRED quality scores it will use. By default it uses the Solexa quals.
Forum: Bioinformatics 10-04-2012, 12:50 PM
Replies: 6
Views: 1,638
Posted By mnkyboy
You can use fastx to filter based on your desired...

You can use fastx to filter based on your desired quality score.
Forum: RNA Sequencing 10-03-2012, 01:07 PM
Replies: 6
Views: 2,352
Posted By mnkyboy
I have done a lot of both in mammalian systems...

I have done a lot of both in mammalian systems and RiboZero is the way to go. We got some great results with DSN when it worked but it can be a little sensitive and the normalization can fail...
Forum: Illumina/Solexa 08-15-2012, 12:08 PM
Replies: 10
Views: 5,195
Posted By mnkyboy
We do a tiny fraction of DNA compared to RNA-seq....

We do a tiny fraction of DNA compared to RNA-seq. The mass conversion has worked for these, but I do take into account the size if they are different from our standard RNA libraries and rely on the...
Forum: Illumina/Solexa 08-15-2012, 09:46 AM
Replies: 10
Views: 5,195
Posted By mnkyboy
I have done a lot of TruSeq RNA and I find that I...

I have done a lot of TruSeq RNA and I find that I can get very standard clustering using mass measurement on the QuBit. This has been my go to for quite a while, I still do qPCR but this mass off...
Forum: Bioinformatics 08-03-2012, 02:13 PM
Replies: 2
Views: 3,448
Posted By mnkyboy
You will have to post-process your SAM file after...

You will have to post-process your SAM file after bowtie:

http://sourceforge.net/apps/mediawiki/samtools/index.php?title=SAM_FAQ
Forum: Bioinformatics 05-15-2012, 08:14 AM
Replies: 4
Views: 5,718
Posted By mnkyboy
I was wondering if you were still going to post...

I was wondering if you were still going to post that script? I was looking for an easy solution for this as well.
Forum: Bioinformatics 05-01-2012, 09:04 AM
Replies: 15
Views: 9,610
Posted By mnkyboy
You will only really be able to find poly...

You will only really be able to find poly adenylated non coding RNAs and possible miRNA precursors.

If you are truly interested in longer non coding you will need Total RNA-seq libraries. For...
Forum: RNA Sequencing 04-27-2012, 09:18 AM
Replies: 21
Views: 11,161
Posted By mnkyboy
Also to echo ETHANol I have used both RiboMinus...

Also to echo ETHANol I have used both RiboMinus and RiboZero and there is not comparison. In fact RiboMinus should not even be an option they don't even touch the performance of the RiboZero beads...
Forum: RNA Sequencing 04-27-2012, 09:14 AM
Replies: 21
Views: 11,161
Posted By mnkyboy
I have done it both ways using Total RNA-seq...

I have done it both ways using Total RNA-seq bypassing the poly-A step with the TruSeq kits.

In my hands the RiboZero works extremely well, but DSN can work just as well but if you are not...
Forum: General 03-29-2012, 01:59 PM
Replies: 23
Views: 6,903
Posted By mnkyboy
Have them get AWS accounts.

Have them get AWS accounts.
Forum: Illumina/Solexa 03-27-2012, 12:15 PM
Replies: 16
Views: 6,777
Posted By mnkyboy
I have played with changing --mismatches to 1 and...

I have played with changing --mismatches to 1 and have not seen a huge gain. Also two of the indexes are not compatible if you do this. If you use GACGAC and CACGAT with mismatch 1 it will fail,...
Forum: RNA Sequencing 03-08-2012, 09:23 AM
Replies: 6
Views: 2,397
Posted By mnkyboy
Try making a ribosomal index and see what...

Try making a ribosomal index and see what percentage of your samples are ribosomal. There may have been an inconsistency in the library prep.

Also what are the samples? For example in some of...
Forum: Sample Prep / Library Generation 03-04-2012, 09:39 AM
Replies: 3
Views: 3,371
Posted By mnkyboy
If it is whole transcriptome it will not matter,...

If it is whole transcriptome it will not matter, if you are doing some mRNA-seq you may see some loss but if it is for assembly you should be fine since you are probably doing a lot of reads.
Forum: Illumina/Solexa 02-15-2012, 12:55 PM
Replies: 5
Views: 5,665
Posted By mnkyboy
Are all of the other libraries higher in...

Are all of the other libraries higher in concentration? I have seen in lower concentration libraries the curve is less rounded and you see individual peaks like you are seeing.

Based on your dye...
Forum: Bioinformatics 02-08-2012, 09:54 AM
Replies: 4
Views: 1,730
Posted By mnkyboy
I use chrome almost exclusively and rarely have a...

I use chrome almost exclusively and rarely have a problem.

Truth is if there is an incompatibility you have to do the unspeakable and use Explorer.
Forum: Bioinformatics 02-07-2012, 08:13 AM
Replies: 10
Views: 5,341
Posted By mnkyboy
You probably have a lot of non miRNA in your...

You probably have a lot of non miRNA in your sequencing. Have you tried mapping just to the genome? We see a ton of non miRNA small RNA in our small RNA sequencing.
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