Adaption for preprocessing methods

Introduction

nanoCEM now supports four alignment methods for nanopore signal: f5c resquiggle, f5c eventalign, move_table from basecaller, and tombo resquiggle.

While tombo resquiggle and f5c eventalign align the signals to the reference sequence, f5c resquiggle and move_table from basecaller are alignment methods applied to the basecalled sequence. Thus,these basecalled sequence methods require additional adaptions to index the reference sequence.

Basecalled sequence methods adaption

Signal index file acquisition

For f5c resquiggle, a paf (Pairwise Alignment Format) file is generated, which records the alignment information and indices corresponding to the basecalled sequence.

On the other hand, the move_table is stored in the bam file generated from the basecaller, for the related decoding and indexing methods, detailed description is here. To address this issue, we utilized the squigualiser reform tool available in the repository here to generate the PAF file.

Re-indexing on the reference sequence

For such methods, we align the basecalled sequence to the reference by using the CIGAR values from the bam file to generate an index table. This process involves discarding insertions and deletions while preserving mismatches to accurately map the basecalled sequence onto the reference.

Base shift (only for f5c)

For f5c (both for resquiggle and eventalign), during nanopore signal alignment, it utilizes a k-mer model and assigns the alignment result to the first base. However, this is not the case for tombo resquiggle and move_table. Their developers noticed this issue and applied a complicated strategy to tackle it in this link.

But in nanoCEM, to make their results as consistent as possible and enable comparisons with tombo and move_table, we introduced --base_shift option to align the result closer to the most contributing base, and shift num is recorded here

The default value for --base_shift is auto. However, if you want to disable it, you can use --base_shift 0. If you want to shift by a different number, please enter a negative value, for example, --base_shift -3. Please note that for RNA mode, the shift will be from 3' to 5', so the value should also be a negative number.