mitoflow

A short and sweet pipeline for assembling mitochondria and filtering reads prior to genome assembly. Intended for use with Illumina paired reads.

Note that in practice, I haven't really found a large difference between assemblies produced with or without filtering mitochondrial reads. That is, not with SPAdes in Parastagonospora nodorum. Given the small amount of mitochondrial sequence genuinely present in the nuclear genome (based on long read assemblies), I play it safe and assemble with the full read set. I then use these assemblies to fish out the mitochondrial contigs using mashmap.

Usage

cat <<EOF > reads.tsv
sample	read1_file	read2_file
one	pair1_R1.fastq.gz	pair1_R2.fastq.gz
one	pair2_R1.fastq.gz	pair2_R2.fastq.gz
two other_R1.fastq.gz	other_R2.fastq.gz
EOF

nextflow run -resume darcyabjones/mitoflow \
  --reference mito.fasta \
  --seed "seeds/*.fasta" \
  --asm_table reads.tsv \
  --filter_table reads.tsv \
  --read_length 150 \
  --insert_size 320

Mandatory Arguments

param                   | description
---------------------------------------------------------------------------
`--asm_table <tsv>`     | A table mapping fastq read pairs to samples
                        | (See Tables section).

`--reference <fasta>`   | A fasta formatted reference mitochondrial
                        | assembly from a closely related isolate.
                        | Multiple files can be provided using glob
                        | patterns.

`--read_length <int>`   | The length of the fastq reads. This can also
                        | be provided in the tsv provided by
                        | `--asm_table` (See Tables section).

`--insert_length <int>` | The insert size of the fastq pairs. This can
                        | also be provided in the tsv provided by
                        |`--asm_table` (See Tables section).

Options

param                  | default     | description
---------------------------------------------------------------------------
`--filter_table <tsv>` | none        | A table of reads to filter with
                       |             | their corresponding samples
                       |             | (See tables section). If not
                       |             | provided will skip read filtering
                       |             | steps.

`--seed <fasta>`       | --reference | Fasta formatted sequences to seed
                       |             | the mitochondrial assembly. This
                       |             | could be a mitochondrial gene or
                       |             | assembly from a closely related
                       |             | isolate. Multiple fasta files can
                       |             | be specified using a glob pattern.

`--min_size <int>`     | 12000       | The minimum size (bp) of the
                       |             | mitochondrial assemblies.

`--max_size <int>`     | 100000      | The maximum size (bp) of the
                       |             | mitochondrial assemblies

`--kmer <int>`         | 39          | The K-mer size (bp) to use for the
                       |             | NOVOplasty assembly

Tables

Because individual samples are often sequenced in multiple runs, and given in multiple fastq pairs, mitoflow takes input to --asm_table and --filter_table as tab separated values (tsv) files. Three columns are mandatory for both tables: sample, read1_file, and read2_file. The column order is not important, but a header line must be included. Filenames should be either absolute paths or relative to the executing path. The sample column is the factor how the fastq pairs will be grouped for assemblies and for deciding which assemblies to filter against. It will also be the base name of the resultant assemblies.

--asm_table can also use two additional columns read_length and insert_size, which will override the --read_length or --insert_size parameters for this sample. Note that if you don't provide --read_length or --insert_size all rows must have corresponding values in these columns. --filter_table can also use the additional column merged_file which can be a single "stitched" fastq file to be filtered separately from the pairs.

The --asm_table and --filter_table can be the same file. Additional columns will be ignored. However, be aware that NOVOplasty can't use merged reads, so don't use your pre-filtered reads for the --asm_table. See the examples folder in the github repo for examples.

Outputs

• assemblies/*_mitochondrial.fasta: The assembled mitochondria per sample.

• assemblies/*_log.txt: Logs from NOVOPlasty for assemblies.

• alignments/*.{delta,mcoords,...}: MUMmer files from alignment between assemblies and reference.

• filtered_reads/<sample>: Filtered fastq pairs named same as input.

• filtered_reads/*_mitochondrial.fastq.gz: Filtered fastq pairs aligning to Mitochondria.

• filtered_reads/*_scafstats.txt: BBSplit statistics containing number of reads aligned to different scaffolds.

• filtered_reads/*_refstats.txt: BBSplit statistics containing number of reads aligned to different references (either --reference or assembly for this sample).

Requirements

• NOVOPlasty https://github.com/ndierckx/NOVOPlasty. Developed with v2.7.2. NB the version in Conda is too old and will give an error about having "no input". You can currently download from https://github.com/ndierckx/NOVOPlasty/raw/master/NOVOPlasty2.7.2.pl, and link it to somewhere on your $PATH as NOVOPlasty.pl.
• MUMmer https://github.com/mummer4/mummer. Developed with v4.0.0beta2.
• BBMap https://sourceforge.net/projects/bbmap/. Developed with v38.39 but bbsplit is generally fairly stable. (Optional, only required if filtering reads)