Table of Contents

• pyScaf
  • Scaffolding modes
    • NGS-based scaffolding
    • Scaffolding based on long reads
    • Reference-based scaffolding
  • Usage
    • Dependencies
    • Parameters
    • Test run
  • Proof of concept

pyScaf

pyScaf orders contigs from genome assemblies utilising several types of information:

• paired-end (PE) and/or mate-pair libraries (NGS-based mode)
• long reads (Scaffolding based on long reads)
• synteny to the genome of some related species (Reference-based scaffolding)

Scaffolding modes

NGS-based scaffolding

This is under development... Stay tuned.

Scaffolding based on long reads

In this mode, pyScaf aligns long reads onto the contigs, identifies the reads the connects two or more contigs and join adjacent contigs.

Long reads are aligned locally onto contigs, ignoring:

• matches not satisfying cut-offs (--identity and --overlap)
• suboptimal matches (only best match of each query to reference is kept)
• and removing overlapping matches on reference.

Note, this is experimental implementation.

Reference-based scaffolding

In reference-based mode, pyScaf uses synteny to the genome of closely related species in order to order contigs and estimate distances between adjacent contigs.

Contigs are aligned locally onto reference chromosomes, ignoring:

• matches not satisfying cut-offs (--identity and --overlap)
• suboptimal matches (only best match of each query to reference is kept)
• and removing overlapping matches on reference.

In preliminary tests, pyScaf performed superbly on simulated heterozygous genomes based on C. parapsilosis (13 Mb; CANPA) and A. thaliana (119 Mb; ARATH) chromosomes, reconstructing correctly all chromosomes always for CANPA and nearly always for ARATH (Figures in dropbox, CANPA table, ARATH table). Runs took ~0.5 min for CANPA on 4 CPUs and ~2 min for ARATH on 16 CPUs.

Important remarks:

• Reduce your assembly before (fasta2homozygous.py) as any redundancy will likely break the synteny.
• pyScaf works better with contigs than scaffolds, as scaffolds are often affected by mis-assemblies (no de novo assembler / scaffolder is perfect...), which breaks synteny.
• pyScaf works very well if divergence between reference genome and assembled contigs is below 20% at nucleotide level.
• pyScaf deals with large rearrangements ie. deletions, insertion, inversions, translocations. Note however, this is experimental implementation!
• Consider closing gaps after scaffolding.

Usage

Dependencies

• LAST v700+
• FastaIndex

Parameters

Given reference genome, the program generates pairwise genome alignment (dotplots) by default.

• Genral options:

  -h, --help	show this help message and exit
  -f FASTA, --fasta FASTA
  	assembly FASTA file
  -o OUTPUT, --output OUTPUT
  	output stream [scaffolds.fa]
  -t THREADS, --threads THREADS
  	max no. of threads to run [4]
  --log LOG	output log to [stderr]
  --dotplot	generate dotplot as [png]
  --version	show program's version number and exit

• Reference-based scaffolding options:

  -r REF, --ref REF, --reference REF
  	reference FastA file
  --identity IDENTITY
  	min. identity [0.33]
  --overlap OVERLAP
  	min. overlap [0.66]
  -g MAXGAP, --maxgap MAXGAP
  	max. distance between adjacent contigs [0.01 * assembly_size]
  --norearrangements
  	high identity mode (rearrangements not allowed)

• Long read-based scaffolding options (EXPERIMENTAL!):

  -n LONGREADS, --longreads LONGREADS
  	FastQ/FastA file(s) with PacBio/ONT reads

• NGS-based scaffolding options (!NOT IMPLEMENTED!):

  -i FASTQ, --fastq FASTQ
  	FASTQ PE/MP files
  -j JOINS, --joins JOINS
  	min pairs to join contigs [5]
  -a LINKRATIO, --linkratio LINKRATIO
  	max link ratio between two best contig pairs [0.7]
  -l LOAD, --load LOAD
  	align subset of reads [0.2]
  -q MAPQ, --mapq MAPQ
  	min mapping quality [10]

Test run

To perform reference-based assembly, provide assembled contigs and reference genome in FastA format. Dotplots of below runs can be found in docs. If you wish to skip dotplot generation (ie. no X11 on your system), provide --dotplot '' parameter.

# scaffold homogenised assembly (reduced contigs)
./pyScaf.py -f test/contigs.reduced.fa -r test/ref.fa -o test/contigs.reduced.ref.fa

# scaffold reduced contigs using global mode (no norearrangements allowed)
./pyScaf.py -f test/contigs.reduced.fa -r test/ref.fa -o test/contigs.reduced.ref.global.fa --norearrangements

# scaffold heterozygous assembly (de novo assembled contigs)
./pyScaf.py -f test/contigs.fa -r test/ref.fa -o test/contigs.ref.fa

# scaffold reduced contigs using long reads
## pacbio
./pyScaf.py -f test/contigs.reduced.fa -n test/pacbio.fq.gz -o test/contigs.reduced.pacbio.fa
## nanopore
./pyScaf.py -f test/contigs.reduced.fa -n test/nanopore.fa.gz -o test/contigs.reduced.nanopore.fa

# generate dotplot
lastdb test/ref.fa
lastal -f TAB test/ref.fa test/contigs.reduced.pacbio.fa | last-dotplot - test/contigs.reduced.pacbio.fa.ref.png
lastal -f TAB test/ref.fa test/contigs.reduced.nanopore.fa | last-dotplot - test/contigs.reduced.nanopore.fa.ref.png

# clean-up
#rm test/contigs.{,reduced.}fa.* test/ref.fa.* test/*.{nanopore,pacbio,ref}* test/*.log

Proof of concept

pyScaf is under heavy development right now. Nevertheless, both the reference-based mode and long-read mode are functional and produces meaningful assemblies. pyScaf has been implemented in Redundans.

For more info, have a look in workbook.