Posts

Get the newest Reactome gene sets for pathway analysis

Image
For first-pass pathway analysis I find Reactome to be the most useful database of gene sets for biologists to understand. For a long time I have been using Reactome gene sets as deposited to the GSEA/MSigDB website. Recently a colleague pointed me to the gene matrix file offered directly on the Reactome webpage (Thanks Dr Okabe).

There are some differences. Firstly there are more gene sets in the one from the Reactome webpage (accessed 2018-05-09)
$ wc -l *gmt     674 c2.cp.reactome.v6.1.symbols.gmt    2022 ReactomePathways.gmt    2696 total
Secondly, there are more genes included in one or more gene sets:
$ cut -f3- c2.cp.reactome.v6.1.symbols.gmt | tr '\t' '\n' | sort -u | wc -l 6025 $ cut -f3- ReactomePathways.gmt | tr '\t' '\n' | sort -u | wc -l 10852
And overall there are about threefold more gene-pathway entries 
$ cut -f3- ReactomePathways.gmt | wc -w 106405 $ cut -f3- c2.cp.reactome.v6.1.symbols.gmt | wc -w 37601
I also looked at whether the gen…

Publishing datasets on the dat network - benefits and pitfalls

Image
As I mentioned in an earlier post, Dat is a new data sharing tool that uses concepts of bittorrent and git to enable peer-to-peer sharing of versioned data. This is cool for sharing datasets that change over time, because when you sync the dataset, only the changes are retrieved, sort of like git. As it uses peer-to-peer technology, it is fairly resilient to node failures as the datasets are mirrored between peers. The "dat publish" command registers the repository on datbase.org, meaning that the files can be retrieved by anyone via a normal browser.

To demonstrate, I have released the bulk data dumps from my RNA-seq data processing project, DEE2, which consists of 158 GB of gene expression data. These data are freely available via a browser at https://datbase.org/dee2/bulk or by using the dat command-line tool.


If you're after a single file, then you can use the following syntax to retrieve over https:

wget https://datbase.org/download/<long dat address>/<file…

Has RNA-seq overtaken microarrays?

Image
We know RNA-seq has a number of advantages over array based analyses, but is RNA-seq taking over in terms of number of datasets published? I got curious and thought I'd investigate with some PubMed searches. I searched for "RNA-seq" and "microarray" and downloaded the CSV file which summarises the number of citations per year. As a type of control, I also searched "gene expression".

I divided the yearly "RNA-seq" and "Microarray" citation counts by the "Gene expression" counts then multiplied by 1000 to give the numbers seen below.

You can see that microarray is still more frequent in PubMed as compared to RNA-seq, but the gap is getting much narrower and the cross will likely occur in the next two years.

Next, I will look at the rate of GEO data deposition. (Updates soon)

Share and backup data sets with Dat

Image
If you work in genomics, you'll know that sharing large data sets is hard. For instance our group has shared data with our collaborators a number of ways:

DVDs, hard drives and flash drivesFTPHightailGoogle Drive linksAmazon linksSCP/PSCPrsync
But none of these are are ideal as we know data sets change over time and none of the above methods are suited to updating a file tree with changes. If changes occur, then it quickly becomes a mess of files that are either redundant or missing entirely. Copied files could become corrupted. What we need is a type of version control for data sets. That's the goal of dat.

So now I'll take you through a simple example of sharing a data set using dat.

#Install instructions for Ubuntu 16.04
$ sudo npm cache clean -f
$ sudo npm install -g n
$ sudo n stable
$ sudo npm install -g dat

# Files I'm sharing on PC 1: DGE table and 3 genelists (3.4 MB)
$ tree
.
├── Aza_DESeq_wCounts.tsv
└── list
    ├── Aza_DESeq_wCounts_bg.txt
    ├── Aza_DESeq_wCounts_dn.…

Update on DEE2 project for Jan 2018

Image
Today I'd like to share some updates on the DEE2 project, which I wrote about  in an earlier post. The  project code can be viewed on github here. Pipeline and images As the pipeline was recently finalised, I was able to roll out the working docker image. To facilitate users without root access, this image was ported to singularity. This took a lot of effort and some expertise from our local HPC team to get things working (many thanks to the Massive/M3 team). The singularity image is available from the webserver (link) and instructions for running it are available on github here. I have started testing a heavyweight singularity image, which includes the genome indexes, which will be more efficient for running jobs with large genomes and will make it available once testing is complete. Queue management It may sound simple to write a script to determine which datasets have been completed and add new datasets to the queue but when taking about tens of thousands of datasets it's …

Update on the DEE project Dec 2017

Back in 2015, our group described DEE, a user friendly repository of uniformly processed RNA-seq data, which I covered in detail in a previous post. Ours was the first such repository that wasn't limited to human or mouse and included sequencing data from a variety of instruments and library types. The purpose of this post is to reflect on the mixed success of DEE and outline where this project is going in future.

Overall I've received a lot of positive feedback from users and a number of citations to our poster. Thanks to everyone who used, gave suggestions, comments, bug reports, etc! However our attempt to have the repository published wasn't so successful due to reviewer niggles over what I consider minor points but hard to implement quickly. The main points raised by reviewers were:

Is it reasonable to treat all data sets as if they were single end? For this one, the reviewers were split, one said it was OK and the other was adamant that it was unacceptable despite my …

Diagnosing PCR duplicates from cluster duplicates

Image
NovaSeq, HiSeqX and HiSeq4000 Illumina sequencers have patterned flowcells which have a different chemistry as compared to random clustered flowcell systems (Hiseq2500 & MiSeq) which is known to cause duplicates during the clustering process. For some background on the issue, see these previous blog posts:

QC Fail blog Steve WingettEnseqlopedia blog by James Hadfield In my recent whole genome bisulfite sequencing experiment using TruSeq methylation library prep kits and NovaSeq, I noticed a high proportion of duplicate reads and wanted to investigate whether these were "cluster" duplicates, ie generated during the clustering process due to ExAmp chemistry or were duplicates generated during the PCR step. Generally cluster duplicates occur in the immediate proximity on the flowcell surface and PCR duplicates are expected to occur uniformly throughout the flowcell surface.
To diagnose this, I used the diagnose-dups tool by Dave Larson which can be found on Github here. I wr…