base_shift_and_eventalignment.md

Base shift calculation

• User can shift the base sequence to the left by n number of bases by providing the argument --base_shift -n to plot and plot_pileup commands.
• This is helpful to correct the signal level to the base. A negative n value will shift the base sequence to the left.
• Base shift concept was motivated from the idea presented in pore_model document about the most contributing base to the current level.
• We can programmatically calculate a base_shift to nicely align the signal to the base (color). The calculation is implemented here.
• However, the user is adviced to use --profile (documented here) which automatically sets the --base_shift.

Example 1

• At site A21 there is a heterozygous variant A/T.

• The first pileup (Fig. 1) has a wrong base shift (-5) and second pileup (Fig. 2) has the correct base shift (-6).

• Hence, the second pileup (Fig. 2) has both the variant (blue box - bed annotation) and the signal differences aligned to each other.

• If the base shift was some other value instead of -6 the difference in the signals will not align with base.

• This variant example is documented here. Figure 1

• Figure 2

Example 2

• Fig. 3 and 4 have the same dna_r10.4.1_e8.2_400bps signal pileup with a base shift of 0 and -6 respectively.
• The base colors in Fig. 3 do not nicely aligned to the signal.
• However, in Fig. 4 the signal is moving from low to high when a base T is met.
• These signal pileups were generated using f5c eventalign.
• F5c used dna_r10.4.1_e8.2_400bps pore model to align these signals. Its most contributing base index is -6 and hence the appropriate base shift in this scenario is also -6.

Figure 3: base shift 0 link

Figure 4: base shift -6 link

Example 3

• Consider a dna_r10.4.1_e8.2_400bps forward and reverse mapped pileups for the same genomic region.
• Fig.5 has 0 base shift for both tracks.
• In Fig. 6, note that the reverse mapped pileup has a -2 base shift. This is because the signal sequencing direction is from right to left (more information).
• In Fig. 6, both pileups have the signals going from low to high when a base T is met.

Figure 5: base shift 0, 0 link

Figure 6: base shift -6, -2 link

Example 4

Fig. 7 shows the forward and reverse mapped pileups generated using f5c eventalign for dna_r9.4.1_450bps data. F5c used the 6mer model (more information).

Figure 7: base shift -2, -3

Example 5

• Fig. 8 shows the forward mapped pileups generated using f5c eventalign for rna_r9.4.1_70bps data. F5c used the rna 5mer model (more information).
• Note that squigualiser always plot the RNA reads in its correct sequencing direction (reverse mapped RNA reads are skipped; reverse mapped RNA reads exist if a genome was used as the reference instead of a transcriptome).

Figure 8: base shift -3