MinION Coverage sensitivity analysis

Introduction

MinION is a new (at time of writing, 2017) sequencing technology capable of very long reads (sometimes, 30kbp).

Due to its lower maturity compared to other technologies, such as Illumina, it also is less robust, leading to scenarios where quality can be too low. In Illumina, such reads are usually discarded, but it in MinION it is less easy to make that decision, because:

• longer reads inherently more valuable due to their ability to span long genomic regions
• too high a quality threshold may discard the majority of reads

While retaining low quality sequencing is not usually a merit of an experiment where high quality reads are in abundance, it is beneficial to at least accompany these with a sensivity analysis which may help extract the usefulness of such reads.

Conversion of base-called fast5 to fasta

This is done with poretools, and can be performed. Filters may be

Subtract Operations

In the spirit of discovering new sequences, this ivolves subtract what is currently annotated from our alignment dataset.

This is a highly untargeted approach because all currently annotated genes and interestung areas are subtracted so that the coverage can only describe currently unknown areas of the genome, including non-coding areas.

Though this might sound unfruitful to start with, it has some benefits:

• useful to gain a feeling of general coverage
• reduces reliance on currently known areas, which may have been chosen becaus eof high coverage from previous studies.

Obtaining the sequence behind a range

As the bed file only holds chromosome names and ranges, there is no sequence information. This is obtain with the bedtools getfasta tool. This however needs a fasta file, so the correct fasta must be obtained from the bam file, because the reference sequence could be used or sequences from the reads.