Alignment

The alignment step maps basecalled reads against a reference genome. If not already done alignment rules will trigger basecalling of the raw nanopore reads.

To align the reads of one flow cell against reference genome hg38 with guppy basecalling and aligner minimap2 run from within your processing directory:

snakemake --snakefile /path/to/nanopype/Snakefile alignments/minimap2/guppy/WA01/batches/20180101_FAH12345_FLO-MIN106_SQK-LSK108_WA01.hg38.bam

This requires an entry hg38 in your env.yaml in the Nanopype installation directory:

references:
    hg38:
        genome: /path/to/references/hg38/hg38.fa
        chr_sizes: /path/to/references/hg38/hg38.chrom.sizes

Providing a runnames.txt with one runname per line it is possible to process multiple flow cells at once and merge the output into a single track e.g.:

snakemake --snakefile /path/to/nanopype/Snakefile alignments/minimap2/guppy/WA01.hg38.bam

Some aligners require an indexed reference genome, Nanopype will automatically build one uppon first use. An alignment job is always connected with a sam2bam conversion and sorting in an additional thread. The above commands with 3 configured alignment threads will therefore fail if Snakemake is invoked with less than 4 threads (at least -j 4 is required). The default input sequence format for alignment rules is fastq, yet if possible, the alignment module will use already basecalled batches in the sequences/[basecaller]/[runname]/ folder of the working directory.

Folder structure

The alignment module can create the following file structure relative to the working directory:

|--alignments/
   |--minimap2/                                                 # Minimap2 alignment
      |--albacore/                                              # Using albacore basecalling
         |--batches/
            |--WA01/
               |--20180101_FAH12345_FLO-MIN106_SQK-LSK108_WA01/
                  |--0.hg38.bam
                  |--1.hg38.bam
                  ...
               |--20180101_FAH12345_FLO-MIN106_SQK-LSK108_WA01.hg38.bam
         |--WA01.hg38.bam                                       # Merged flow-cells
         |--WA01.hg38.bw                                        # Coverage track
   |--graphmap2/                                                 # GraphMap2 alignment
      |--guppy/                                                 # Using guppy basecalling
         |--batches/
            |--WA01/
               |--20180101_FAH12345_FLO-MIN106_SQK-LSK108_WA01/
                  |--0.hg19.bam
                  ...
               |--20180101_FAH12345_FLO-MIN106_SQK-LSK108_WA01.hg19.bam
         |--WA01.hg19.bam
         |--WA01.hg19.bw

Cleanup

The batch processing output of the alignment module can be cleaned up by running:

snakemake --snakefile /path/to/nanopype/Snakefile alignment_clean

This will delete all files and folders inside of any batches directory and should only be used at the very end of an analysis workflow. The methylation module for instance relies on the single batch alignment files.

Tools

Depending on the application you can choose from one of the listed aligners. All alignment rules share a set of global config variables:

• threads_alignment: 3

Minimap2

Pairwise alignment for nucleotide sequences. Any given command line arguments are directly passed to the aligner:

• alignment_minimap2_flags: '-ax map-ont -L'

GraphMap2

Fast and sensitive mapping of nanopore sequencing reads with GraphMap2. Any given command line arguments are directly passed to the aligner:

• alignment_graphmap2_flags: '-B 100'

NGMLR

Accurate detection of complex structural variations using single-molecule sequencing. Any given command line arguments are directly passed to the aligner:

• alignment_ngmlr_flags: '-x ont --bam-fix'

References

Li, H. Minimap2: pairwise alignment for nucleotide sequences. Bioinformatics. doi:10.1093/bioinformatics/bty191 (2018).

Sovic, I. et al. Fast and sensitive mapping of nanopore sequencing reads with GraphMap. Nat. Commun. 7:11307 doi: 10.1038/ncomms11307 (2016).

Sović, I., Šikić, M., Wilm, A. et al. Fast and sensitive mapping of nanopore sequencing reads with GraphMap. Nat Commun 7, 11307 (2016)

Sedlazeck, F. J. et al. Accurate detection of complex structural variations using single-molecule sequencing. Nature Methods 15, 461-468 (2018).