FPKM, RPKM and TPM

We've discussed these gene expression metrics a few times recently, so below is a video that gives a nice explanation for each of them. https://www.youtube.com/watch?v=TTUrtCY2k-w Also of interest is one of the comments, asking about comparisons across samples that aren't replicates. A small quote from the reply: To be honest, TPM, FPKM and RPKM etc … Continue reading FPKM, RPKM and TPM →

RNA extraction – Trizol protocol

EDIT: no sooner do I publish this than I discover that there is actually a kit that combines Trizol extraction and the PureLink columns. We've got a couple of these Trizol protocols hanging around the lab, just thought it might be a good idea to save them here for posterity. Trizol extraction is good for … Continue reading RNA extraction – Trizol protocol →

RNA-seq – how many replicates?

I think we've discussed this paper before in lab meetings, but just thought I'd pop it here for posterity. How many biological replicates are needed in an RNA-seq experiment and which differential expression tool should you use? The authors performed an RNA-seq experiment with 48 replicates per condition, identified the significantly differentially expressed genes, and … Continue reading RNA-seq – how many replicates? →

On the optimal use of methylKit

This is a useful paper from 2017 titled Strategies for analyzing bisulfite sequencing data. It reviews sequencing and alignment approaches, general methylome analysis, differential methylation and annotation, as well as recommending analysis platforms for non-coders. Section 4.1 discusses differential methylation methods, comparing three different methods using a simulated dataset, including as a subset of one method … Continue reading On the optimal use of methylKit →

Notifications for new blog posts

If you follow the blog you'll get notifications to let you know about new posts - by default this takes the form of an email every time someone posts something. You can control the frequency of the notifications by going here while logged in and changing the settings for this blog. You can switch between … Continue reading Notifications for new blog posts →

BS-seq problems – the solution

In the previous post I described a problem with BS-seq data. After much messing about I found the solution in the analysis tool methylKit. Rather than using the final output of the Bismark aligner tool (the 'coverage' or 'cytosineReport' files) you can use methylKit to process the BAM file Bismark makes in order to generate a file … Continue reading BS-seq problems – the solution →

Bisulfite sequencing – bad tiles on the flowcell

Recently I've had a great deal of difficulty with some BS-seq data, which had some bad reads where the quality dropped off randomly in the middle of the sequence - this is unusual as quality issues are typically at the ends of reads. You can see this illustrated below. The poor quality reads seem to … Continue reading Bisulfite sequencing – bad tiles on the flowcell →