Analysis with PAUSE3 – 2016 Edition

How to Analyze Your Data with PAUSE3

Once you’ve run your analysis in PAUSE, you may be wondering how to interpret the plots you receive. This short guide should assist you in just that.

Good Results

After running PAUSE Plotter on raw sequencing data, good quality data will produce output that looks like the following:

Selection_558

Here the “Full depth” is just the sequencing coverage of every base in the genome. It gives us a region of interest to zoom in on.

 Selection_559

This looks somewhat like what you’re probably used to!

The “full coverage” track corresponds to both the sense + antisense mapped reads merged together, then we just display the read start tracks as the read-end tracks did not provide useful data in PAUSE2.

moonbeam

With a good quality start, we can even zoom down to the individual base level and see exactly where the reads start to pile up:

Selection_560

Here we can see a spike to 58 on the sense track, which corresponds to base 282 in the genome.

Bad Results

palmer

Data sets will appear noisy and you will find no defined sense or antisense starts. In truth, the read start data is no more noisy than in good quality data, however due to the much lower sequencing depth, the spikes are significantly higher relatively to the sequence coverage plots (gray). In this case it is not possible to see pile-ups

There are endless possible failure modes with no interpretation, take for example the following:

dirt8

In other cases, you will see a gradual pile-up of starts or ends and will not be able to confidently predict either the sense/antisense start/end to the terminal repeat. In these cases, terminal redundancies cannot be predicted. Experimentation or re-sequencing will be needed to determine genomic ends or terminal redundancy boundaries, respectively.

Quick Guide

Type Description
Long TRs Characterised by regions of double coverage >1.5kb
Short TRs Characterised by regions of double coverage 0-1kb
Non-Cos/Headful packaging strategies No defined appearance in PAUSE and require PCR for closure.

Future Work

We’ll be bringing back the automated peak calling hopefully soon! Stay tuned for updates.