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Processing_with_linked_reads.md

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1. RagTag

Before processing with raw linked-read. We used the Supernova produced assembly to scaffold the assembly. We used RagTag for that purpurse.

Script to run RagTag

#!/bin/bash -e

#SBATCH --nodes 1
#SBATCH --cpus-per-task 1
#SBATCH --ntasks 10
#SBATCH --job-name Ragtag.EW
#SBATCH --mem=10G
#SBATCH --time=05:00:00
#SBATCH --account=uoo02752
#SBATCH --output=%x_%j.out
#SBATCH --error=%x_%j.err
#SBATCH --mail-type=ALL
#SBATCH [email protected]
#SBATCH --hint=nomultithread

export PATH="/path/to/the/installation/directory/of/RagTag:$PATH"  # Installation path, conda installation available

ragtag.py scaffold \
                path/to/the/supernova/assembly/EW_10xSN.fasta \
                path/to/the/gapclosed/assembly/after/LR_Gapcloser/assembly.fasta

2.ARBitR

We used raw linked read data to scaffold the assembly with ARBitR Before that, we had to process the assembly file and map linked-reads to the processed assembly using 'Longranger' Scripts here. The resulting Bam file was used as input for ARBitR.

Script to run ARBitR

#!/bin/bash -e

#SBATCH --nodes 1
#SBATCH --cpus-per-task 1
#SBATCH --ntasks 10
#SBATCH --job-name Arbitr.ew
#SBATCH --mem=5G
#SBATCH --time=05:00:00
#SBATCH --account=uoo02752
#SBATCH --output=%x_%j.out
#SBATCH --error=%x_%j.err
#SBATCH --mail-type=ALL
#SBATCH [email protected]
#SBATCH --hint=nomultithread

export PATH="/path/to/installation/directory/of/ARBitR/src:$PATH"
export PATH="/path/to/conda/environment/where/dependencies/are/installed:$PATH" # dependencies were installed in conda environment

arbitr.py -i ragtag.scaffold.fasta \                                    # This is the output from the RagTag above
          -o output.arbitr.scaffolds \
          path/to/bam/file/from/longranger/outs/possorted_bam.bam      # Bamfile produced by longranger-align

3. Arks & Links

We further used Arks pipeline to scaffold the assembly. We had to process the linked read with longranger-basics to prepare an input Scripts here

Script to run Arks pipeline

#!/bin/bash -e

#SBATCH --nodes 1
#SBATCH --cpus-per-task 1
#SBATCH --ntasks 16
#SBATCH --job-name arks.EW
#SBATCH --mem=25G
#SBATCH --time=18:00:00
#SBATCH --account=uoo02752
#SBATCH --output=%x_%j.out
#SBATCH --error=%x_%j.err
#SBATCH --mail-type=ALL
#SBATCH [email protected]
#SBATCH --hint=nomultithread

module load BWA/0.7.17-GCC-9.2.0
module load SAMtools/1.13-GCC-9.2.0
module load BEDTools/2.29.2-GCC-9.2.0
module load LINKS/1.8.7-GCC-9.2.0

export PATH="/Path/to/installation/directory/arks-1.0.4/Arks:$PATH"
export PATH="/Path/to/installation/directory/arks-1.0.4/Examples:$PATH"

arks-make arks draft=assembly \    # assembly file to scaffold should be in working directory (or symlink) should have the name supplied to draft with .fasta extension
                reads=barcoded \    # barcoded.fastq.gz should be in the working directory (or symlink)
                threads=16