Basecalling process carried out by full_1dsq_basecaller.py/.exe

Question

I am unable to understand what exactly the full_1dsq_basecaller.py/.exe script does. Is it giving the consensus sequence for linear and complementary strand, or it is just giving the reads that came from the complimentary strand?

If we are getting two fastq files as output which one we should use for alignment?

Thank you in advance. I greatly appreciate any help

Answer 1

My understanding is that the 1D$^2$ process is carried out in three stages:

1. Carry out a 1D calling of reads (i.e. what the standard Albacore does at the moment)
2. Link reads that are similar to each other (in reverse-complement orientation), from the same channel, and have a very short pore transition time
3. Create a 2D consensus base call, using information from the two linked reads

The two outputs from the basecaller are the 1D calls from step 1, and the consensus calls from step 3. Ideally just the 2D consensus calls should be used for assembly (assuming it represents a random distribution of reads), but if that gives too low coverage (or is not random enough), then the non-converted reads from the 1D calls could be included as well.

Because the linking is not a physical link, it's not a good idea to use 1D$^2$ for amplicon reads; the likelihood of two complementary reads from different templates appearing one after another is too high.