...

One consequence of using multithreading that might be confusing is that the aligned reads might appear in your output SAM file in a different order than they were in the input FASTQ. This happens because small sets of reads get continuously packaged, "sent" to the different processors, and whichever set "returns" fastest is written first. You can force them to appear in the same order (at a slight cost in speed) by adding the --reorder flag to your command, but is typically only necessary if the reads are already ordered or you intend to do some comparison between the input and output. 

What comes after mapping?

The next steps are often to view the output using a specific viewer on your local machine, or to begin identifying variant locations where the reads differ from the reference sequence. These will be the next things we cover in the course.

Optional exercises 

• In the bowtie2 example, we mapped in --local mode. Try mapping in --end-to-end mode (aka global mode).

• Do the BWA tutorial so you can compare their outputs.
  • Did bowtie2 or BWA map more reads?
  • In our examples, we mapped in paired-end mode. Try to figure out how to map the reads in single-end mode and create this output.
  • Which aligner took less time to run? Are there any options you can change that:
    • Lead to a larger percentage of the reads being mapped? (increase sensitivity)
    • Speed up run time without causing many fewer reads to be mapped? (increase performance)

Next steps...

The next steps are often to view the output using a specific viewer on your local machine, or to begin identifying variant locations where the reads differ from the reference sequence. These will be the next things we cover in the course. Here is a link to help you return to the GVA 2020 course schedule.