# Per Base Sequence Content

## Summary

Per Base Sequence Content plots out the proportion of each base position in a file for which each of the four normal DNA bases has been called.

In a random library you would expect that there would be little to no difference between the different bases of a sequence run, so the lines in this plot should run parallel with each other. The relative amount of each base should reflect the overall amount of these bases in your genome, but in any case they should not be hugely imbalanced from each other.

It's worth noting that some types of library will always produce biased sequence composition, normally at the start of the read. Libraries produced by priming using random hexamers (including nearly all RNA-Seq libraries) and those which were fragmented using transposases inherit an intrinsic bias in the positions at which reads start. This bias does not concern an absolute sequence, but instead provides enrichement of a number of different K-mers at the 5' end of the reads. Whilst this is a true technical bias, it isn't something which can be corrected by trimming and in most cases doesn't seem to adversely affect the downstream analysis. It will however produce a warning or error in this module.

## Warning

This module issues a warning if the difference between A and T, or G and C is greater than 10% in any position.

## Failure

This module will fail if the difference between A and T, or G and C is greater than 20% in any position.

## Common reasons for warnings

There are a number of common scenarios which would ellicit a warning or error from this module.

1. Overrepresented sequences: If there is any evidence of overrepresented sequences such as adapter dimers or rRNA in a sample then these sequences may bias the overall composition and their sequence will emerge from this plot.
2. Biased fragmentation: Any library which is generated based on the ligation of random hexamers or through tagmentation should theoretically have good diversity through the sequence, but experience has shown that these libraries always have a selection bias in around the first 12bp of each run. This is due to a biased selection of random primers, but doesn't represent any individually biased sequences. Nearly all RNA-Seq libraries will fail this module because of this bias, but this is not a problem which can be fixed by processing, and it doesn't seem to adversely affect the ablity to measure expression.
3. Biased composition libraries: Some libraries are inherently biased in their sequence composition. The most obvious example would be a library which has been treated with sodium bisulphite which will then have converted most of the cytosines to thymines, meaning that the base composition will be almost devoid of cytosines and will thus trigger an error, despite this being entirely normal for that type of library
4. If you are analysing a library which has been aggressivley adapter trimmed then you will naturally introduce a composition bias at the end of the reads as sequences which happen to match short stretches of adapter are removed, leaving only sequences which do not match. Sudden deviations in composition at the end of libraries which have undergone aggressive trimming are therefore likely to be spurious.