The move table format used by ONT basecallers to store the move information is not generalised. For instance, a move that is not a multiple of the stride, cannot be stored in the move table format. A more generalised format to store the signal to read/reference alignment is documented here, which we would refer to as the ss tag. In addition to the flexibility, this ss tag format has a greater compression ratio (~1.6) in ASCII format compared to the move table format.
Squigualiser uses this format ss tag as the input to generate plots. Hence, before proceeding to create plots, the move table has to be converted to the required ss tag format. This is done using the subtool reform in squigualiser.
The two important parameters reform take are kmer_length (or k) and sig_move_offset (or m).
This determines the base colour adjustment to the signal jumps. More information is discussed in pore_model document and using base shift.
The user can provide --profile argument to determine the kmer length and the signal move offset using preset values.
Optionally the user can also provide -k and -m values. The argument --profile will override -k and/or -m values, if all arguments are provided.
ALIGNMENT=reform_output.paf
PROFILE="guppy_dna_r10.4.1_e8.2_400bps_fast"
squigualiser reform -c --bam basecalls.sam -o ${ALIGNMENT} --profile ${PROFILE}
KMER_LENGTH=1
SIG_MOVE_OFFSET=0
squigualiser reform -k ${KMER_LENGTH} -m ${SIG_MOVE_OFFSET} -c --bam basecalls.sam -o ${ALIGNMENT}
Precomputed kmer lengths and signal move offsets can be found here.
If reform errors out with the message "Error: specified profile is not found. Please run reform with -k 1 -s 0. Then run calculate_offsets.py and rerun reform with the recommended kmer_length and sig_move_offset.", then the user is advised to run calculate_offsets subtool .
Information regarding calcualte_offsets is documented at calculate_offsets.
The ASCII BYTE count ratio between the two formats can be easily calculated using the following bash commands.
BASECALLER_MOVES_FILE=basecalls.sam
REFORM_FILE=reform_output.paf
mv_array_size=$(samtools view ${BASECALLER_MOVES_FILE} | awk '{for(i=5;i<=NF;i++){if($i~/mv/){a=$i}} print a}' | wc -c)
ss_array_size=$(cat ${REFORM_FILE} | awk '{for(i=5;i<=NF;i++){if($i~/ss:Z:/){a=$i}} print a}' | wc -c)
awk -v var1=${mv_array_size} -v var2=${ss_array_size} 'BEGIN { print ( var1 / var2 ) }'
Reform can also output a custom TSV format for human readability (not supported for plotting):
squigualiser reform -k ${KMER_LENGTH} -m ${SIG_MOVE_OFFSET} --bam basecalls.sam -o reform_output.tsv