Gap-Free Long-read Assembler (GALA)

GALA is a Gap-free Long-read Assembler. GALA builds a multi-layer graph from different preliminary assemblies, long-reads, and potentially other sources of information, such as Hi-C assemblies. During this process, it identifies mis-assembled contigs and trim them. The corrected data are then partitioned into multiple scaffolding groups, each representing a single chromosome. Each scaffolding group is assembled independently with existing assembly tools and a simplified version of overlap-graph-based merging algorithm is used to merge multiple contigs if necessary.

GALA has three modules each can be used separately.

GALA performance with a human genome

GALA assembled a human genome using (HiFi) reads. GALA used canu draft for CHM13 and the current human reference genome GRCh38.p13 as input of GALA. In this way GALA essentially created a reference-guided de novo assembly. GALA assembly comprised of 37 continuous contigs, including 8 telomer-to-telomer gap-free pseudomolecular sequences, 4 near complete chromosomes each with a small telomeric fragment unanchored, 3 with only gapped centromeric regions, and the long arm of acrocentric chromosomes. Human Genome

Dependency

1. Minimap2
2. bwa
3. samtools
4. python2.7
5. canu

Installation

GALA can be run directly from the gala folder

git clone https://github.com/ganlab/gala.git cd GALA

Or You can run install to add it to your PATH

Usage

Using GALA pipeline to assemble a genome involves preliminary steps and three main Steps.

Preliminary step and Inputs

Preliminary step

Use different software to construct preliminary assemblies from long reads, e.g. (Canu, Flye, MECAT, Miniasm, and Wtdbg2).

Inputs:

• Raw reads and corrected reads if available.
• The user needs to prepare draft_names_paths.txt for preliminary assemblies. Here is an example:

draft_01=path/to/draft_fasta_file
draft_02=path/to/draft_fasta_file
draft_03=path/to/draft_fasta_file
draft_n=path/to/draftfasta file

GALA Single Command Mode

To run GALA using one command user can use the following command:

gala draft_names_paths.txt fa/fq reads_file platform

In single command mode, GALA used canu for Chromosome-by-Chromosome assembly.

To use another assembler or multiple assemblers, GALA provides three choices Canu, Flye, and Miniasm, pass it to -a argument with a single space between them.

For sequencing_platform the user needs to provide it in this way: -pacbio-raw -pacbio-corrected -nanopore-raw -nanopore-corrected

usage: gala -h  [options] <draft_names & paths> <fa/fq> <reads> <platform>

GALA Gap-free Long-read Assembler

positional arguments:
  draft_names           Draft names and paths [required]
  input_file            input type (fq/fa) [required]
  reads                 raw/corrected reads [required]
  sequencing_platform   -pacbio-raw -pacbio-corrected -nanopore-raw -nanopore-
                        corrected [required]

optional arguments:
  -h, --help            show this help message and exit
  -a [ASSEMBLER [ASSEMBLER ...]]
                        Chr-by_Chr assembler (canu flye miniasm) [default
                        canu]
  -b Alignment block length	 [default 5000]
  -p Alignment identity percentage	 [default 70%]
  -c Shortest contig length	 [default 5000]
  -q Mapping quality	 [default 20]
  -f Output files name	[default gathering]
  -o output files path	[default current directory]
  -v, --version         show program's version number and exit

GALA Step-by-Step Mode (recommended)

Mis Assembly Detector Module (MDM)

1. Use the comp module to generate a draft_comparison file

   comp draft_names_paths.txt

2. Run draft_comparison file to produce drafts comparison paf files

   sh draft_compare.sh

3. Use the mdm module to identify mis-assembled contigs.

   mdm comparison_folder number of assembly drafts

4. Use the newgenome module to Produce misassembly-free drafts.

   newgenome draft_names_paths.txt cut_folder

Contig Clustering Module (CCM)

5. Use the comp module to generate a draft_comparison file for misassembly-free drafts.

   comp new_draft_names_paths.txt

6. Run draft_comparison file to produce new drafts comparison paf files.

   sh draft_compare.sh

7. Run the ccm module to produce contigs scaffolding groups.

   ccm comparison_folder number of assembly drafts

   • Note: You can also use the reformat module to generate reformatted paf files and use them to confirm Scaffolding groups.

Scaffolding Group Assembly Module (SGAM)

8. Map all drafts against raw long reads and self-corrected reads if available.

   bwa index misassembly-free draft bwa mem -x pacbio/ont2d misassembly-free draft long-reads

9. Use the following commands to separate the read names mapped to each contig

   samtools view -H bam_file |grep "SQ"|cut -f 2|cut -d : -f 2 > contig_names

   seprator contig_names mapping.bam

   sh bam_seprator.sh

   for i in bams/*; do samtools view $i | cut -f 1 > $i.read_names;done;

10. Use the cat command to concatenate read name files belongs to the same scaffolding group.

    • For example:

      cat contig_1.bam.read_names contig_3.bam.read_names contig_7.bam.read_names > scaffold_1.read_names

11. Use the readsep Module to separate each scaffold correlated-reads.

    for i in scaffold_*.read_names; do readsep raw/correted-reads $i -f input reads file type fa/fq

12. Implement Chromosome-by-Chromosome assembly approach to retrieve the gap-free chromosome-scale assembly by

    Assemble each read set from scaffold_*.read.fq with different
    assembly software, e.g.(Canu, Flye, Mecat, Miniasm, and Wtdbg).
    

    we recommend the user to try different assembly tools especially ( Flye, MECAT/NECAT, and Miniasm)

13. Finally, map the SGAM outcomes against one of the preliminary draft assemblies to confirm that all the contigs in the scaffolding group are assembled to the right chromosome/Scaffold.

Description

comp:

The comp module used to generate a genome comparison file if the user wants to compare multiple genomes against each other.

usage: comp -h  [options] <draft_names & paths> 

Generate genome comparison files, part of GALA Gap-free Long-read Assembler

positional arguments:
  drafts                Draft names and paths [required]

optional arguments:
  -h, --help            show this help message and exit
  -o output files path	[default current directory]
  -v, --version         show program's version number and exit

mdm:

Miss-assembly Detector Module used to detect misassembled contigs. The algorithm relies on the alignment's contradictory information.

mis-assembly detection module should be applicable for error correction regardless of the specific algorithm used for assembly and can differentiate between misassembly and Structure variation

usage: mdm -h  [options] path/to/mapping_files number of drafts
MDM Mis-assembly Detector Module, part of GALA Gap-free Long-read Assembler

positional arguments:
  mapping_files         mapping paf file [required]
  drafts                Number of drafts [required]

optional arguments:
  -h, --help            show this help message and exit
  -b Alignment block length	 [default 5000]
  -p Alignment identity percentage	 [default 70%]
  -c Shortest contig length	 [default 5000]
  -q Mapping quality	 [default 20]
  -f Output files name	[default gathering]
  -o output files path	[default current directory]
  -v, --version         show program's version number and exit

newgenome:

The newgenome module trims the misassembled contigs and gives misassembly free genome. This module used only with multiple samples

usage: newgenome -h  [options] <draft_names & paths> <path to cut files>

Produce mis-assembly free genomes, part of GALA Gap-free Long-read Assembler 

positional arguments:
  draft                 Draft names and paths [required]
  cut_files             path_to_cut_files" [required]

optional arguments:
  -h, --help            show this help message and exit
  -f Output files name	[default new_genome]
  -o output files path	[default current directory]
  -v, --version         show program's version number and exit

ccm:

Contig Clustering Module used to identify the scaffolding groups and the contigs overlap information in multiple preliminary assemblies.

ccm could have extended applications in generating consensus assembly from multiple sequences. Besides, it is useful in reference guide scaffolding to determine Chromosomes scaffolding groups

usage: ccm -h  [options] <path/to/mapping_files> <number of drafts>

CCM Contig Clustering Module, part of GALA Gap-free Long-read Assembler

positional arguments:
mapping_files         mapping paf file [required]
drafts                Number of drafts [required]

optional arguments:
-h, --help            show this help message and exit
-b Alignment block length	 [default 5000]
-p Alignment identity percentage	 [default 70%]
-c Shortest contig length	 [default 5000]
-q Mapping quality	 [default 20]
-f Output files name	[default scaffolds]
-o output files path	[default current directory]
-v, --version         show program's version number and exit

reformat

the reformat module filters the alignment data in paf mapping files and merge overlapping and continuous alignment intervals into a single mapping interval. So, each contig in query draft will have one alignment interval with the subject draft.

usage: reformat -h  [options] <path/to/mapping_files> <number of drafts>

Re-formatting mapping files module, part of GALA Gap-free Long-read Assembler

positional arguments:
  mapping_files         mapping paf file [required]
  drafts                Number of drafts [required]

optional arguments:
  -h, --help            show this help message and exit
  -b Alignment block length	 [default 5000]
  -p Alignment identity percentage	 [default 70%]
  -c Shortest contig length	 [default 5000]
  -q Mapping quality	 [default 20]
  -f Output files name	[default reformated]
  -o output files path	[default current directory]
  -v, --version         show program's version number and exit

seprator

The seprator module used to separate contigs alignments in individual bams and separate the read names mapped to each contig in an individual file

usage: seprator -h  [options] <contig_names> <bam_file>

Separate each contig correlated read names, part of GALA Gap-free Long-read Assembler

positional arguments:
  contig_names          contig_names [required]
  bam_file              mapping bam file [required]

optional arguments:
  -h, --help            show this help message and exit
  -o output files path	[default current directory]
  -f Output files name	[default bam_seprator]
  -b output folder name	 [default bams]
  -v, --version         show program's version number and exit

Use the following command to produce contig_names file:
	samtools view -H <bam_file> |grep 'SQ'|cut -f 2|cut -d : -f 2 > contig_names

readsep

The readsep module separates a set of reads from a sequencing dataset according to the read name in the definition line.

usage: readsep -h [options] <reads> <read_titles>

Extract reads from fasta or fastq, part of GALA Gap-free Long-read Assembler

positional arguments:
  reads                 raw/corrected reads [required]
  read_titles           read names [required]

optional arguments:
  -h, --help            show this help message and exit
  -f input file format (fa/fq)
  -v, --version         show program's version number and exit

Licence

GALA is distributed under MIT license. See the LICENSE file for details.