Sequence alignment

The output of the sequencing run is a fastq file with sequences and other additional information such as quality scores and coordinates. All these sequences have to be aligned to the input sequence(s) because there might be some insertions, deletions or mismatches. If multiple samples are present, this step is also to determine which sequence matches which sample. The output of sequence alignment is a sam file, which is needed for the step where the single-molecule and sequencing data are linked.

We suggest using Bowtie 2 for the sequence alignment. All files needed for sequence alignment can be found in the sequence_analysis folder. If this is the first time, run the Sequencing_setup file to create the right environment. More detailed instructions on for example how to run Linux on Windows can be found in the Analysis.sh file in the sequence_analysis folder.

Some of the Bowtie 2 options:

--local

In this mode, Bowtie 2 does not require that the entire read aligns from one end to the other. Rather, some characters may be omitted (“soft clipped”) from the ends in order to achieve the greatest possible alignment score.

--np

Sets penalty for positions where the read, reference, or both, contain an ambiguous character such as N.

--very-sensitive-local

Preset with: -D 20 -R 3 -N 0 -L 20 -i S,1,0.50

--n-ceil

Sets a function governing the maximum number of ambiguous characters (e.g. N) allowed in a read as a function of read length. For instance, L,0,1 gives f(x) = 0 + 1 * x, where x is the read length.

--threads

By default bowtie2-build is using only one thread. Increasing the number of threads will speed up the index building considerably in most cases.

--score-min

Sets a function governing the minimum alignment score needed for an alignment to be considered “valid” (i.e. good enough to report). Here G,20,8 gives f(x) = 20 + 8 * log(x), where x is the read length.

--norc

With this setting, Bowtie 2 will not attempt to align unpaired reads against the reverse-complement (Crick) reference strand.

-L

Sets the length of the seed substrings to align during multiseed alignment. Smaller values make alignment slower but more sensitive. Default 20 in –very-sensitive-local mode. For very short reads with a stretch of N’s in the middle, lower this setting.

--reorder

Makes the order of sequences in the sam file similar to the original fastq file. This is important when using multiple threads and indexes need to be added later on.

Check the manual of Bowtie2 for an explanation of all the options: https://bowtie-bio.sourceforge.net/bowtie2/manual.shtml