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Forum: Sample Prep / Library Generation 01-10-2019, 12:42 PM
Replies: 0
Views: 431
Posted By rflrob
Low-concentration, AT rich genome sequencing

Hi all,
Wondering what the current consensus is on recommended protocols for gDNA library prep from small concentrations (1-5ng) of an AT rich genome (Dictyostelium discoideum, ~23% GC). ...
Forum: Bioinformatics 05-08-2015, 03:13 PM
Replies: 3
Views: 1,246
Posted By rflrob
I'm not familiar with this "do" module you seem...

I'm not familiar with this "do" module you seem to be using, but I'm pretty sure that passing a list is the equivalent of spaces on the command line. For instance, in the original cuffdiff call,...
Forum: Bioinformatics 05-07-2015, 03:51 PM
Replies: 3
Views: 1,246
Posted By rflrob
It looks to me like you're perhaps missing a...

It looks to me like you're perhaps missing a comma after cfg.project["samples"][0]["files"]["bam"]. As a general debugging strategy, when you have massive blocks of code like that, it's often easier...
Forum: Bioinformatics 01-30-2014, 03:20 PM
Replies: 9
Views: 1,330
Posted By rflrob
You might try: In bash or similar: $ ftp ...

You might try:

In bash or similar:
$ ftp

# now in FTP
> open ftp.ncbi.nlm.nih.gov
> cd blast/executables/blast+/2.2.28
> ls
> get APPROPRIATE_FILE_HERE
Forum: RNA Sequencing 01-15-2014, 03:59 PM
Replies: 0
Views: 840
Posted By rflrob
Superscript "Stitching" low complexity fragments?

Hi all,
I have a low complexity RNA sample (think a purified pool of a single gene) that I'm trying to prepare for sequencing using the TruSeq kit, and I'm ending up with large (typically >1kb)...
Forum: Bioinformatics 09-27-2013, 09:03 AM
Replies: 1
Views: 869
Posted By rflrob
You might try checking out miRBase:...

You might try checking out miRBase: http://www.mirbase.org/
Forum: Bioinformatics 09-13-2013, 10:48 AM
Replies: 3
Views: 3,682
Posted By rflrob
This snippet of python might do the trick for...

This snippet of python might do the trick for you, for the simplest possible definition of "counting mismatches", where that's any column where it's not all identical. It assumes you have BioPython...
Forum: Bioinformatics 09-13-2013, 09:12 AM
Replies: 7
Views: 2,568
Posted By rflrob
I've done a similar thing in flies, pooling, then...

I've done a similar thing in flies, pooling, then disambiguating reads: http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0071820

The strategy I used was to make a combined...
Forum: RNA Sequencing 06-17-2013, 02:46 PM
Replies: 1
Views: 2,209
Posted By rflrob
It's almost certainly that way so that if you...

It's almost certainly that way so that if you write something to deal with a fpkm_tracking file, you'll always know that it has the same number of columns. The generic format is described here:...
Forum: Bioinformatics 06-07-2013, 11:50 AM
Replies: 1
Views: 2,221
Posted By rflrob
Null pointer exception in Java TreeView

Hi all,
I'm trying to K-means cluster some data using Cluster 3.0, then view it using Java TreeView (assorted links to software here http://bonsai.hgc.jp/~mdehoon/software/cluster/software.htm), but...
Forum: RNA Sequencing 05-22-2013, 02:38 PM
Replies: 3
Views: 1,119
Posted By rflrob
We do find that we pick up reads that map to lots...

We do find that we pick up reads that map to lots of these non-coding RNAs, but that they actually throw off the FPKM calculations quite a bit. I remove anything that I don't trust from my GTF file...
Forum: Bioinformatics 05-09-2013, 11:13 AM
Replies: 5
Views: 2,086
Posted By rflrob
How many reads do you have? I'm not certain how...

How many reads do you have? I'm not certain how many cufflinks wants before it will estimate them, but it's somewhere under a million.
Forum: Bioinformatics 05-06-2013, 01:11 PM
Replies: 4
Views: 3,549
Posted By rflrob
Cufflinks not masking features in the mask GTF file?

Hi all,
I'm running cufflinks with the mask option:
cufflinks --num-threads 50 --output-dir analysis-multi/CaS3A -u -M Reference/AAA/suspect.gtf --frag-bias-correct Reference/AAA/mel_only.fa ...
Forum: Bioinformatics 04-15-2013, 10:19 AM
Replies: 7
Views: 2,226
Posted By rflrob
If I recall correctly, you don't need to paste...

If I recall correctly, you don't need to paste them together.

The md5 files are checksums. If you're worried that the file didn't download properly, you can run the md5 program on your own...
Forum: Literature Watch 02-21-2013, 05:10 PM
Replies: 0
Views: 1,102
Posted By rflrob
Sequencing mRNA from cryo-sliced embryos to determine genome-wide spatial expression

Complex spatial and temporal patterns of gene expression underlie embryo differentiation, yet methods do not yet exist for the efficient genome-wide determination of spatial patterns of gene...
Forum: Bioinformatics 02-18-2013, 03:00 PM
Replies: 7
Views: 1,902
Posted By rflrob
Even if you don't have one line per gene, you can...

Even if you don't have one line per gene, you can still use the same trick of opening the handles once:


handle1 = open(...)
handle2 = open(...)

for gene in handle1:
# do stuff
for...
Forum: Bioinformatics 02-15-2013, 10:33 AM
Replies: 14
Views: 2,364
Posted By rflrob
Another nice thing about parallel is that it...

Another nice thing about parallel is that it makes it easy to generate filenames in an intelligent way. Say you want to convert a bunch of bam files to sam files, you can easily do:

parallel...
Forum: Bioinformatics 02-06-2013, 05:40 PM
Replies: 4
Views: 1,347
Posted By rflrob
Not sure if Bioinformatics is the right place for...

Not sure if Bioinformatics is the right place for this question, given that there's an Illumina section, but I'm pretty sure both ends do need an adapter to do the bridge PCR. If you're doing...
Forum: Bioinformatics 01-15-2013, 10:06 AM
Replies: 17
Views: 6,381
Posted By rflrob
Sounds to me like Siva's probably got the right...

Sounds to me like Siva's probably got the right idea, but it's also worth pointing out that tophat will, by default, sort the reads in accepted_hits. You can turn this off, but if you're just going...
Forum: Bioinformatics 01-10-2013, 10:58 AM
Replies: 3
Views: 1,651
Posted By rflrob
Talking Cufflinks "GffObj::getSpliced() error: improper genomic coordinate"

Cufflinks is giving me this error:

$ cufflinks -p 4 -o analysis-multi/CaS1A/ -u -N -b Reference/AAA/mel_only.fa -G Reference/AAA/mel_only.gtf analysis-multi/CaS1A/assigned_dmel_rescued.bam ...
Forum: Bioinformatics 01-03-2013, 02:15 PM
Replies: 2
Views: 1,445
Posted By rflrob
If you have a fasta file with those 25 genes, you...

If you have a fasta file with those 25 genes, you can then run bowtie-build on that, and then use the resulting index for bowtie or tophat.
Forum: Bioinformatics 12-29-2012, 04:35 PM
Replies: 2
Views: 961
Posted By rflrob
If t1, t2, and t3 are really just the same sample...

If t1, t2, and t3 are really just the same sample that got sequenced in multiple lanes, then it's more correct to do:
tophat -o [output] -G [gff] reference...
Forum: Bioinformatics 12-28-2012, 10:59 AM
Replies: 2
Views: 1,196
Posted By rflrob
Read.h is a source code file (specifically, a...

Read.h is a source code file (specifically, a header file). When I download quake, there's both a "bin" directory (containing BINary executables), and a "src" directory, which is where Read.h is. ...
Forum: Bioinformatics 12-26-2012, 01:54 PM
Replies: 1
Views: 1,173
Posted By rflrob
Cuffdiff reports FPKMs outside the range of replicates that go into it

Hi all,
I'm having an issue where cuffdiff is reporting an FPKM value for the sample is greater than any of the FPKMs reported for the 3 replicates. I'm pretty sure I'm running everything with the...
Forum: Bioinformatics 12-13-2012, 03:05 PM
Replies: 2
Views: 1,035
Posted By rflrob
I think it makes sense that the different paired...

I think it makes sense that the different paired ends will be on opposite strands (since you basically read the second end forwards, but on the other strand). At the level of zoom you're at in the...
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