The repository contains all the scripts that I used for assembling the genome of the clover root weevil( Sitona obsoletus) using data from a combination of long read nanopore, 10x genomics and Illumina sequencing technology. The long read assembly from nanopore MinION was used as a primary assembly which is then sccafolded and gap closed using short Illumina reads and 10x linked reads. Schematic representation of my assembly pipeline is given below;
Raw data (fast5) from oxford nanopore MinION was basecalled using Guppy version 5. The script for Guppy is given below.
Script for Guppy 5
#!/bin/bash -e
#SBATCH --job-name=guppy_crw #name of the job
#SBATCH --account=uoo02772 #my project number in nesi
#SBATCH --time=10:00:00 #wall time
#SBATCH --partition=gpu #guppy runs faster in gpu partition in nesi, than other partition
#SBATCH --gres=gpu:1 #some configuration for gpu partition
#SBATCH --mem=6G # memory 6gb
#SBATCH --ntasks=4 #ntask set to 4
#SBATCH --cpus-per-task=1 #cpu per task set to 1
#SBATCH --output=%x-%j.out #%x gives job name and %j gives job number, this is slurm output file
#SBATCH --error=%x-%j.err #similar slurm error file
#SBATCH --mail-type=ALL
#SBATCH --mail-user=katma889@student.otago.ac.nz
module load ont-guppy-gpu/5.0.7
guppy_basecaller -i ../ -s . --flowcell FLO-MIN106 --kit SQK-LSK109 --num_callers 4 -x auto --recursive --trim_barcodes --disable_qscore_filtering
Guppy provided us with the merged fastqc files along with the sequencing summary.txt file as an input which we process further with pycoQC-2.5.2 to check the quality of our data. It resulted in an interactive html file with details of data quality.
Script for pycoqc
#!/bin/bash -e
#SBATCH --nodes 1
#SBATCH --cpus-per-task 1
#SBATCH --ntasks 10
#SBATCH --partition=large
#SBATCH --job-name pycoqc
#SBATCH --mem=50G
#SBATCH --time=01:00:00
#SBATCH --account=uoo02772
#SBATCH --output=%x.%j.out
#SBATCH --error=%x.%j.err
#SBATCH --mail-type=ALL
#SBATCH --mail-user=katma889@student.otago.ac.nz
#SBATCH --hint=nomultithread
export PATH="/nesi/nobackup/path/to/bin/miniconda3/bin:$PATH"
pycoQC -f ../sequencing_summary.txt -o pycoQC_output.html
After viwing the quality of our output data via html file we further proceed to remove the reads mapping to the lambda phage genome from our fastq files using 'Nanolyse'. This is because we used DNA CS while running our sample in the minion flow cells. The script for 'Nanolyse' is given below which was used to remove lambda DNA from our fastq files.
Script for Nanolyse
#!/bin/bash -e
#SBATCH --nodes 1
#SBATCH --cpus-per-task 1
#SBATCH --ntasks 10
#SBATCH --partition=large
#SBATCH --job-name nanolyse.job
#SBATCH --mem=50G
#SBATCH --time=08:00:00
#SBATCH --account=uoo02752
#SBATCH --output=%x.%j.out
#SBATCH --error=%x.%j.err
#SBATCH --mail-type=ALL
#SBATCH --mail-user=katma889@student.otago.ac.nz
#SBATCH --hint=nomultithread
export PATH="/nesi/nobackup/uoo02752/nematode/bin/miniconda3/bin:$PATH"
cat ../crw.ont.merged.fastq | NanoLyse --reference ./dna_cs.fasta | gzip > crw_nanopore_filtered.fastq.gz
Similarly the dna_cs. fasta we used in our experiment is given below;
DNA_CS
GCCATCAGATTGTGTTTGTTAGTCGCTTTTTTTTTTTGGAATTTTTTTTTTGGAATTTTTTTTTTGCGCTAACAACCTCCTGCCGTTTTGCCCGTGCATATCGGTCACGAACAAATCTGATTACTAAACACAGTAGCCTGGATTTGTTCTATCAGTAATCGACCTTATTCCTAATTAAATAGAGCAAATCCCCTTATTGGGGGTAAGACATGAAGATGCCAGAAAAACATGACCTGTTGGCCGCCATTCTCGCGGCAAAGGAACAAGGCATCGGGGCAATCCTTGCGTTTGCAATGGCGTACCTTCGCGGCAGATATAATGGCGGTGCGTTTACAAAAACAGTAATCGACGCAACGATGTGCGCCATTATCGCCTAGTTCATTCGTGACCTTCTCGACTTCGCCGGACTAAGTAGCAATCTCGCTTATATAACGAGCGTGTTTATCGGCTACATCGGTACTGACTCGATTGGTTCGCTTATCAAACGCTTCGCTGCTAAAAAAGCCGGAGTAGAAGATGGTAGAAATCAATAATCAACGTAAGGCGTTCCTCGATATGCTGGCGTGGTCGGAGGGAACTGATAACGGACGTCAGAAAACCAGAAATCATGGTTATGACGTCATTGTAGGCGGAGAGCTATTTACTGATTACTCCGATCACCCTCGCAAACTTGTCACGCTAAACCCAAAACTCAAATCAACAGGCGCCGGACGCTACCAGCTTCTTTCCCGTTGGTGGGATGCCTACCGCAAGCAGCTTGGCCTGAAAGACTTCTCTCCGAAAAGTCAGGACGCTGTGGCATTGCAGCAGATTAAGGAGCGTGGCGCTTTACCTATGATTGATCGTGGTGATATCCGTCAGGCAATCGACCGTTGCAGCAATATCTGGGCTTCACTGCCGGGCGCTGGTTATGGTCAGTTCGAGCATAAGGCTGACAGCCTGATTGCAAAATTCAAAGAAGCGGGCGGAACGGTCAGAGAGATTGATGTATGAGCAGAGTCACCGCGATTATCTCCGCTCTGGTTATCTGCATCATCGTCTGCCTGTCATGGGCTGTTAATCATTACCGTGATAACGCCATTACCTACAAAGCCCAGCGCGACAAAAATGCCAGAGAACTGAAGCTGGCGAACGCGGCAATTACTGACATGCAGATGCGTCAGCGTGATGTTGCTGCGCTCGATGCAAAATACACGAAGGAGTTAGCTGATGCTAAAGCTGAAAATGATGCTCTGCGTGATGATGTTGCCGCTGGTCGTCGTCGGTTGCACATCAAAGCAGTCTGTCAGTCAGTGCGTGAAGCCACCACCGCCTCCGGCGTGGATAATGCAGCCTCCCCCCGACTGGCAGACACCGCTGAACGGGATTATTTCACCCTCAGAGAGAGGCTGATCACTATGCAAAAACAACTGGAAGGAACCCAGAAGTATATTAATGAGCAGTGCAGATAGAGTTGCCCATATCGATGGGCAACTCATGCAATTATTGTGAGCAATACACACGCGCTTCCAGCGGAGTATAAATGCCTAAAGTAATAAAACCGAGCAATCCATTTACGAATGTTTGCTGGGTTTCTGTTTTAACAACATTTTCTGCGCCGCCACAAATTTTGGCTGCATCGACAGTTTTCTTCTGCCCAATTCCAGAAACGAAGAAATGATGGGTGATGGTTTCCTTTGGTGCTACTGCTGCCGGTTTGTTTTGAACAGTAAACGTCTGTTGAGCACATCCTGTAATAAGCAGGGCCAGCGCAGTAGCGAGTAGCATTTTTTTCATGGTGTTATTCCCGATGCTTTTTGAAGTTCGCAGAATCGTATGTGTAGAAAATTAAACAAACCCTAAACAATGAGTTGAAATTTCATATTGTTAATATTTATTAATGTATGTCAGGTGCGATGAATCGTCATTGTATTCCCGGATTAACTATGTCCACAGCCCTGACGGGGAACTTCTCTGCGGGAGTGTCCGGGAATAATTAAAACGATGCACACAGGGTTTAGCGCGTACACGTATTGCATTATGCCAACGCCCCGGTGCTGACACGGAAGAAACCGGACGTTATGATTTAGCGTGGAAAGATTTGTGTAGTGTTCTGAATGCTCTCAGTAAATAGTAATGAATTATCAAAGGTATAGTAATATCTTTTATGTTCATGGATATTTGTAACCCATCGGAAAACTCCTGCTTTAGCAAGATTTTCCCTGTATTGCTGAAATGTGATTTCTCTTGATTTCAACCTATCATAGGACGTTTCTATAAGATGCGTGTTTCTTGAGAATTTAACATTTACAACCTTTTTAAGTCCTTTTATTAACACGGTGTTATCGTTTTCTAACACGATGTGAATATTATCTGTGGCTAGATAGTAAATATAATGTGAGACGTTGTGACGTTTTAGTTCAGAATAAAACAATTCACAGTCTAAATCTTTTCGCACTTGATCGAATATTTCTTTAAAAATGGCAACCTGAGCCATTGGTAAAACCTTCCATGTGATACGAGGGCGCGTAGTTTGCATTATCGTTTTTATCGTTTCAATCTGGTCTGACCTCCTTGTGTTTTGTTGATGATTTATGTCAAATATTAGGAATGTTTTCACTTAATAGTATTGGTTGCGTAACAAAGTGCGGTCCTGCTGGCATTCTGGAGGGAAATACAACCGACAGATGTATGTAAGGCCAACGTGCTCAAATCTTCATACAGAAAGATTTGAAGTAATATTTTAACCGCTAGATGAAGAGCAAGCGCATGGAGCGACAAAATGAATAAAGAACAATCTGCTGATGATCCCTCCGTGGATCTGATTCGTGTAAAAAATATGCTTAATAGCACCATTTCTATGAGTTACCCTGATGTTGTAATTGCATGTATAGAACATAAGGTGTCTCTGGAAGCATTCAGAGCAATTGAGGCAGCGTTGGTGAAGCACGATAATAATATGAAGGATTATTCCCTGGTGGTTGACTGATCACCATAACTGCTAATCATTCAAACTATTTAGTCTGTGACAGAGCCAACACGCAGTCTGTCACTGTCAGGAAAGTGGTAAAACTGCAACTCAATTACTGCAATGCCCTCGTAATTAAGTGAATTTACAATATCGTCCTGTTCGGAGGGAAGAACGCGGGATGTTCATTCTTCATCACTTTTAATTGATGTATATGCTCTCTTTTCTGACGTTAGTCTCCGACGGCAGGCTTCAATGACCCAGGCTGAGAAATTCCCGGACCCTTTTTGCTCAAGAGCGATGTTAATTTGTTCAATCATTTGGTTAGGAAAGCGGATGTTGCGGGTTGTTGTTCTGCGGGTTCTGTTCTTCGTTGACATGAGGTTGCCCCGTATTCAGTGTCGCTGATTTGTATTGTCTGAAGTTGTTTTTACGTTAAGTTGATGCAGATCAATTAATACGATACCTGCGTCATAATTGATTATTTGACGTGGTTTGATGGCCTCCACGCACGTTGTGATATGTAGATGATAATCATTATCACTTTACGGGTCCTTTCCGGTGAAAAAAAAGGTACCAAAAAAAACATCGTCGTGAGTAGTGAACCGTAAGC
As basecalling with Guppy only resulted in removal of adapters attached to the end of the reads. Therefore, 'Porechop' was used to remove any remaining adapters present in the middle of our reads. The scrpit for 'Porechop' is given below;
Script for Porechop
#!/bin/bash -e
#SBATCH --nodes 1
#SBATCH --cpus-per-task 1
#SBATCH --ntasks 10
#SBATCH --partition=large
#SBATCH --job-name porechop
#SBATCH --mem=50G
#SBATCH --time=04:00:00
#SBATCH --account=uoo02772
#SBATCH --output=%x.%j.out
#SBATCH --error=%x.%j.err
#SBATCH --mail-type=ALL
#SBATCH --mail-user=bhaup057@student.otago.ac.nz
#SBATCH --hint=nomultithread
module load Porechop/0.2.4-gimkl-2020a-Python-3.8.2
porechop -i ../crw_nanopore_filtered.fastq.gz -o crw_ont_nanolyse_porechop.fastq.gz --threads 10
Among different assemblers that were tried for assefor Nanopore Long reads,FLye gave the best assembly, Therefore We used FLye2.9 version to assemble the long read data from Oxford Minion. The script for flye assembly algorithm is given below;
Script for Flye
#!/bin/bash -e
#SBATCH --nodes 1
#SBATCH --cpus-per-task 1
#SBATCH --ntasks 10
#SBATCH --partition=hugemem
#SBATCH --job-name flye.crwV3
#SBATCH --mem=150G
#SBATCH --time=72:00:00
#SBATCH --account=uoo02772
#SBATCH --output=%x_%j.out
#SBATCH --error=%x_%j.err
#SBATCH --mail-type=ALL
#SBATCH --mail-user=katma889@student.otago.ac.nz
#SBATCH --hint=nomultithread
module load Flye/2.9-gimkl-2020a-Python-3.8.2
flye --nano-hq ../crw_ont_nanolyse_porechop_nanofilt.fastq.gz -o ./Flye -t 10 -i 3
This Flye output also includes a main file assembly.fasta which was further used for running the Quast. Quast is usually used to evaluate the assembly quality even in the absence of a reference genome. The script of Quast is given below;
Script for Quast
#!/bin/bash -e
#SBATCH --nodes 1
#SBATCH --cpus-per-task 1
#SBATCH --ntasks 6
#SBATCH --partition=large
#SBATCH --job-name quast.crw
#SBATCH --mem=30G
#SBATCH --time=09:00:00
#SBATCH --account=uoo02772
#SBATCH --output=%x_%j.out
#SBATCH --error=%x_%j.err
#SBATCH --mail-type=ALL
#SBATCH --mail-user=katma889@student.otago.ac.nz
#SBATCH --hint=nomultithread
module load QUAST
#=========> running quast for assembly quality
quast.py -t 10 --eukaryote --large --conserved-genes-finding --k-mer-stats \
assembly.fasta \
-o quast
By running the above script it yielded a quast folder which yielded a main file called 'report.txt'.
The above mentioned 'report.txt' yielded us the Complete BUSCO and partial BUSCO percentage and also the number of contigs along with other assembly statistics.
Then we used Purgehaplotigs to remove the haplotigs from our assembly. It helps us to to identify and reassign the duplicate contigs to improve our assembly. The script that we ran is given below;
Script for Purgehaplotigs
#!/bin/bash -e
#SBATCH --nodes 1
#SBATCH --cpus-per-task 1
#SBATCH --ntasks 10
#SBATCH --job-name purgehap.crw
#SBATCH --mem=80G
#SBATCH --time=24:00:00
#SBATCH --account=uoo02772
#SBATCH --output=%x_%j.out
#SBATCH --error=%x_%j.err
#SBATCH --mail-type=ALL
#SBATCH --mail-user=katma889@student.otago.ac.nz
#SBATCH --hint=nomultithread
module load SAMtools/1.12-GCC-9.2.0
module load minimap2/2.20-GCC-9.2.0
module load BEDTools/2.29.2-GCC-9.2.0
export PATH="/nesi/nobackup/uoo02772/bin/miniconda3/bin:$PATH"
#step 1
minimap2 -t 10 -ax map-ont assembly.fasta crw_ont_nanolyse_porechop_nanofilt.fastq.gz \
--secondary=no | samtools sort -m 5G -o aligned.bam -T tmp.ali
#step 2
purge_haplotigs hist -b aligned.bam -g assembly.fasta -t 10
#step 3
purge_haplotigs cov -i aligned.bam.gencov -l 0 -m 20 -h 199 -o coverage_stats.csv
#step 4
purge_haplotigs purge -g assembly.fasta -c coverage_stats.csv -b aligned.bam
#step 5
awk '{print $1",s,"}' gapclosed.fasta.pilon3.fasta.fai > cov_stat.csv
#step 6
purge_haplotigs purge -g gapclosed.fasta.pilon3.fasta -c cov_stat.csv -b aligned.bam
This yielded us the file called curated.fasta which we further ran quast on it. This purge haplotigs helped to reduce the number of contigs with slight decrease in complete BUSCO percent. Therefore we further used the RagTag algorithm a toolset for automating assembly scaffolding and patching our long read assembly. The script for RAgTag is given below;
Script for RagTag
#!/bin/bash -e
#SBATCH --nodes 1
#SBATCH --cpus-per-task 1
#SBATCH --ntasks 10
#SBATCH --job-name ragtag.crw
#SBATCH --mem=8G
#SBATCH --time=04:00:00
#SBATCH --account=uoo02772
#SBATCH --output=%x_%j.out
#SBATCH --error=%x_%j.err
#SBATCH --mail-type=ALL
#SBATCH --mail-user=katma889@student.otago.ac.nz
#SBATCH --hint=nomultithread
export PATH="/nesi/nobackup/uoo02772/path to/bin/miniconda3/bin:$PATH"
ragtag.py scaffold curated.haplotigs.fasta curated.fasta
By running above script we got ragtag.scaffold.fasta as our main output and we can check the stats file ( for example ragtag.scaffold.stats) to see the number of scaffold it removed. Our result is given below;
We further ran 'Quast' in the output file from ragtag that is ragtag.scaffold.fasta and it further reduced to number of contigs.
Then we used lrscaff to further scaffold the assembly from ragtag using long reads that is crw_ont_nanolyse_porechop_nanofilt.fasta in our case. The script for lrscaff is given below;
Script for LRScaff
#!/bin/bash -e
#SBATCH --nodes 1
#SBATCH --cpus-per-task 1
#SBATCH --ntasks 10
#SBATCH --job-name lrscaf.crw
#SBATCH --mem=80G
#SBATCH --time=72:00:00
#SBATCH --account=uoo02772
#SBATCH --output=%x_%j.out
#SBATCH --error=%x_%j.err
#SBATCH --mail-type=ALL
#SBATCH --mail-user=katma889@student.otago.ac.nz
#SBATCH --hint=nomultithread
module load minimap2
#step 1
minimap2 -t 10 ragtag.scaffold.fasta crw_ont_nanolyse_porechop_nanofilt.fasta > ./aln.mm
#step 2
export PATH="/nesi/nobackup/uoo02752/bin/lrscaf/target/:$PATH"
java -Xms80g -Xmx80g -jar /nesi/nobackup/uoo02752/bin/lrscaf/target/LRScaf-1.1.11.jar --contig ragtag.scaffold.fasta --alignedFile aln.mm -t mm -p 10 --output ./scaffolds1
We checked the output from the lrscaff from quast and scaffolds5_scaffolds resulted in the further reduction in the number of contigs.
We further scaffold our genome usimng long DNA sequences from ONT crw_ont_nanolyse_porechop_nanofilt.fasta using RAILS v1.5.1 and Cobbler v0.6.1 Here RAIlS is known as all-in -one scaffolder and gap-filler whereas Cobbler is tool that patch gaps automatically. The script used for this is given below;
Script for RAILS and Cobbler
#!/bin/bash -e
#SBATCH --nodes 1
#SBATCH --cpus-per-task 1
#SBATCH --ntasks 10
#SBATCH --job-name rails.cob.crw
#SBATCH --mem=50G
#SBATCH --time=72:00:00
#SBATCH --account=uoo02772
#SBATCH --output=%x_%j.out
#SBATCH --error=%x_%j.err
#SBATCH --mail-type=ALL
#SBATCH --mail-user=katma889@student.otago.ac.nz
#SBATCH --hint=nomultithread
module load Perl/5.30.1-GCC-9.2.0
module load minimap2
module load SAMtools/1.13-GCC-9.2.0
export PATH="/nesi/nobackup/uoo02752/nematode/bin/RAILS/bin:$PATH"
sh runRAILSminimapSTREAM.sh scaffolds.fasta crw_ont_nanolyse_porechop_nanofilt.fasta 250 0.80 500 2 ont \
/scale_wlg_persistent/filesets/opt_nesi/CS400_centos7_bdw/SAMtools/1.13-GCC-9.2.0/bin/samtools 10
By running the script given above first we will get the output of cobbler as crw_ont_nanolyse_porechop_nanofilt.fasta_vs_scaffolds.fasta_250_0.80_gapsFill.fa in my case which was further used by RAILS to get the final output as crw_ont_nanolyse_porechop_nanofilt.fasta_vs_scaffolds.fasta_250_0.80_rails.scaffolds.fa at the end.
*0 bp gaps are not counted towards the average
crw_ont_nanolyse_porechop_nanofilt.fasta_vs_scaffolds.fasta_250_0.80_rails.log
We then ran Quast for the output file from the rails to evaluate the assembly quality parameters.
Then we further used the LRScaff to further boost the contiguity of our assemly using MinION long filetered reads.The script for LRScaff is given below;
script for LRScaff
#!/bin/bash -e
#SBATCH --nodes 1
#SBATCH --cpus-per-task 1
#SBATCH --ntasks 16
#SBATCH --job-name lr-gapEW
#SBATCH --mem=40G
#SBATCH --time=50:00:00
#SBATCH --account=uoo02752
#SBATCH --output=%x_%j.out
#SBATCH --error=%x_%j.err
#SBATCH --mail-type=ALL
#SBATCH --mail-user=katma889@student.otago.ac.nz
#SBATCH --hint=nomultithread
module load BWA/0.7.17-gimkl-2017a
export PATH=/nesi/nobackup/uoo02752/bin/LR_Gapcloser/src/:$PATH
sh LR_Gapcloser.sh -i crw.scaffolds.fasta -l crw_ont_nanolyse_porechop_nanofilt.fasta -s n -t 16 -r 10
By running above script we got iteration -1 to iteration-10 folders with the common filename gapclosed.fasta in each of them. Then we ran quast (iteration10) to check the quality of the assembly, LRScaff reduced gaps (N's per 100 kbp) in our case using crw_ont_nanolyse_porechop_nanofilt.fasta for gap filling.
We further scaffold the output gapclosed.fasta by our supernova assembly from 10X scaffold crw.10x.all.pseudo.fasta from ragtag.
Script for ragtag
#!/bin/bash -e
#SBATCH --nodes 1
#SBATCH --cpus-per-task 1
#SBATCH --ntasks 10
#SBATCH --job-name ragtag.10x
#SBATCH --mem=50G
#SBATCH --time=05:00:00
#SBATCH --account=uoo02772
#SBATCH --output=%x_%j.out
#SBATCH --error=%x_%j.err
#SBATCH --mail-type=ALL
#SBATCH --mail-user=katma889@student.otago.ac.nz
#SBATCH --hint=nomultithread
export PATH="/nesi/nobackup/uoo02752/nematode/bin/miniconda3/bin:$PATH"
ragtag.py scaffold crw.10x.all.pseudo.fasta gapclosed.fasta
By running above script we got ragtag.scaffold.fasta as output file which we further used for scafollding agian using ragtag. We renamed this output as assembly.fasta and also changed the sequence header to make it compatible to use in scaffolding using ragtag. The script for ragtag for second times is same except using assembly.fasta instead of gapclosed.fasta. By running the ragtag script this time we used ragtag.scaffold.fasta
We further used ARBitR for further merging and scaffolding our current genome assembly from ragtag. As it takes alignment file in the bam/sam format (for example possorted_bam.bam in our case) with 10X Chromium barcodes when provided with genome fasta file used for mapping, it will sort and merge the provided contigs into scaffolds. Therefore, first we created a reference data ragtag.scaffold.fasta and alignment in the folder id CRW using longranger to be used by the ARBitR.
Script for longranger
#!/bin/bash -e
#SBATCH --nodes 1
#SBATCH --cpus-per-task 1
#SBATCH --ntasks 10
#SBATCH --job-name longR_CRW
#SBATCH --mem=50G
#SBATCH --time=72:00:00
#SBATCH --account=uoo02772
#SBATCH --output=%x_%j.out
#SBATCH --error=%x_%j.err
#SBATCH --mail-type=ALL
#SBATCH --mail-user=katma889@student.otago.ac.nz
#SBATCH --hint=nomultithread
export PATH=/nesi/project/uoo02752/bin/longranger-2.2.2:$PATH
#longranger mkref ragtag.scaffold.fasta
longranger align --id=CRW \
--fastqs=/nesi/nobackup/uoo02772/crw/10x/1.raw.hiseq.novaseq \
--reference=path/to/refdata-ragtag.scaffold
Then we ran ARBitR on the aligned scaffold fasta file using possorted_bam.bam.
Script for ARBitR
#!/bin/bash -e
#SBATCH --nodes 1
#SBATCH --cpus-per-task 1
#SBATCH --ntasks 10
#SBATCH --job-name arbitr.crw
#SBATCH --mem=5G
#SBATCH --time=05:00:00
#SBATCH --account=uoo02772
#SBATCH --output=%x_%j.out
#SBATCH --error=%x_%j.err
#SBATCH --mail-type=ALL
#SBATCH --mail-user=katma889@student.otago.ac.nz
#SBATCH --hint=nomultithread
export PATH="/nesi/nobackup/uoo02752/nematode/bin/ARBitR/src:$PATH"
export PATH="/nesi/nobackup/uoo02752/nematode/bin/miniconda3/bin:$PATH"
arbitr.py -i ragtag.scaffold.fasta -o output.arbitr.scaffolds ./CRW/outs/possorted_bam.bam
We further ran arks on the output from the above script.
Script for arks
#!/bin/bash -e
#SBATCH --nodes 1
#SBATCH --cpus-per-task 1
#SBATCH --ntasks 16
#SBATCH --job-name arks.crw
#SBATCH --mem=50G
#SBATCH --time=72:00:00
##SBATCH --time=00:15:00
#SBATCH --account=uoo02772
#SBATCH --output=%x_%j.out
#SBATCH --error=%x_%j.err
#SBATCH --mail-type=ALL
#SBATCH --mail-user=katma889@student.otago.ac.nz
#SBATCH --hint=nomultithread
module load BWA/0.7.17-GCC-9.2.0
module load SAMtools/1.13-GCC-9.2.0
module load BEDTools/2.29.2-GCC-9.2.0
module load LINKS/1.8.7-GCC-9.2.0
export PATH=/path/to/arks-1.0.4/Examples:$PATH
export PATH=/path/to/arks-1.0.4/Arks:$PATH
arks-make arks draft=output.arbitr.scaffolds reads=barcoded threads=16
Then we used Rascafto improve the assembly from above using our PE RNA-seq data. This will enable us to imrove our long-range contiguity and order information from intron-spanning RNA-seq read pairs to improve our draft assembly particularly in gene regions. Therefore we imorted merged fq files for Our PE mRNA-seq reads (merge.R1.fq and merge.R2.fq . Before running the Rascaf we first need to align our RNA-seq reads mapping into our genome using Hisat2.
Script for Hisat2
#!/bin/bash -e
#SBATCH --job-name=hisat.mRNA.crw
#SBATCH --account=uoo02772
#SBATCH --nodes 1
#SBATCH --cpus-per-task 1
#SBATCH --ntasks 10
#SBATCH --mem=40G
#SBATCH --time=20:00:00
#SBATCH --output=%x.%j.out
#SBATCH --error=%x.%j.err
#SBATCH --mail-type=All
#SBATCH --mail-user=katma889@student.otago.ac.nz
#SBATCH --hint=nomultithread
module load HISAT2/2.2.1-gimkl-2020a
module load Python/3.9.5-gimkl-2020a
module load SAMtools/1.13-GCC-9.2.0
hisat2-build -p 10 output.arbitr.scaffolds_c4_m50-10000_k100_r0.05_e30000_z1000_l2_a0.9.scaffolds.fa$
mkdir alignment
hisat2 -x hisat2.RNA.crw -1 ./crw-merged-RNAseq/merge.R1.fq -2 ./crw-merged-RNAseq/merge.R2.fq -S ali$
samtools view -bS alignment/crw_mRNA_alignment.sam > alignment/crw_mRNA_alignment.bam
samtools sort alignment/crw_mRNA_alignment.bam -o alignment/crw_mRNA_alignment_sorted.bam
This gave us the result in SAM file which is then converted to BAM which is further converted into sorted BAM as the final output to be use in our further assembly process using Rescaf.
Script for rascaf
#!/bin/bash -e
#SBATCH --job-name=rascaf.crw
#SBATCH --account=uoo02772
#SBATCH --nodes 1
#SBATCH --cpus-per-task 1
#SBATCH --ntasks 10
#SBATCH --mem=5G
#SBATCH --time=08:00:00
#SBATCH --output=%x.%j.out
#SBATCH --error=%x.%j.err
#SBATCH --mail-type=All
#SBATCH --mail-user=katma889@student.otago.ac.nz
#SBATCH --hint=nomultithread
export PATH=/nesi/nobackup/uoo02752/nematode/bin/rascaf:$PATH
rascaf -b ../crw_mRNA_alignment_sorted.bam \
-f ../../output.arbitr.scaffolds_c4_m50-10000_k100_r0.05_e30000_z1000_l2_a0.9.scaffolds.fa \
-o crw_mRNA_scaffold
rascaf-join -r crw_mRNA_scaffold.out -o crw_mRNA_scaffold
We ran 'Quast' and then Busco version 5.2.2 using insect dataset to evaluate the assembly quality.
After finalising our raw assembly the next step is to re-ran purge-haplotigs using the scripts beow;
Script for purge-haplotigs
#!/bin/bash -e
#SBATCH --nodes 1
#SBATCH --cpus-per-task 1
#SBATCH --ntasks 10
#SBATCH --job-name purgehap.crw
#SBATCH --mem=50G
#SBATCH --time=05:00:00
#SBATCH --account=uoo02752
#SBATCH --output=%x_%j.out
#SBATCH --error=%x_%j.err
#SBATCH --mail-type=ALL
#SBATCH --mail-user=katma889@student.otago.ac.nz
#SBATCH --hint=nomultithread
module load SAMtools/1.13-GCC-9.2.0
module load minimap2/2.20-GCC-9.2.0
module load BEDTools/2.29.2-GCC-9.2.0
#step 1
minimap2 -t 10 -ax map-ont crw_mRNA_scaffold.fa crw_ont_nanolyse_porechop_nanofilt.fasta \
--secondary=no | samtools sort -m 5G -o aligned.bam -T tmp.ali
#step 2
export PATH="/nesi/nobackup/uoo02752/.conda/envs/purge_haplotigs_env/bin:$PATH"
purge_haplotigs hist -b aligned.bam -g crw_mRNA_scaffold.fa -t 10
#step 3
purge_haplotigs cov -i aligned.bam.gencov -l 0 -m 15 -h 190 -o coverage_stats.csv
#step 4
purge_haplotigs purge -g crw_mRNA_scaffold.fa -c coverage_stats.csv -b aligned.bam
#step 5
purge_haplotigs clip -p curated.fasta -h curated.haplotigs.fasta -t 10
Then we further use the output of above script curated.haplotigs.fasta to scaffold our final genome curated.fasta using ragtag.
Script for ragtag
#!/bin/bash -e
#SBATCH --nodes 1
#SBATCH --cpus-per-task 1
#SBATCH --ntasks 10
#SBATCH --job-name ragtag.crw
#SBATCH --mem=50G
#SBATCH --time=05:00:00
#SBATCH --account=uoo02772
#SBATCH --output=%x_%j.out
#SBATCH --error=%x_%j.err
#SBATCH --mail-type=ALL
#SBATCH --mail-user=katma889@student.otago.ac.nz
#SBATCH --hint=nomultithread
export PATH="/nesi/nobackup/uoo02752/nematode/bin/miniconda3/bin:$PATH"
ragtag.py scaffold curated.haplotigs.fasta curated.fasta
By running script above we got the oupur folder ragtag_output with the assembly ragtag.scaffold.fasta which we used for repeating the ragtag assembly for better output named as ragtag2 where the output above is ussed as input for ragtag 2.
Script for 'ragtag2
#!/bin/bash -e
#SBATCH --nodes 1
#SBATCH --cpus-per-task 1
#SBATCH --ntasks 10
#SBATCH --job-name ragtag.crw
#SBATCH --mem=50G
#SBATCH --time=05:00:00
#SBATCH --account=uoo02772
#SBATCH --output=%x_%j.out
#SBATCH --error=%x_%j.err
#SBATCH --mail-type=ALL
#SBATCH --mail-user=katma889@student.otago.ac.nz
#SBATCH --hint=nomultithread
export PATH="/nesi/nobackup/uoo02752/nematode/bin/miniconda3/bin:$PATH"
ragtag.py scaffold curated.haplotigs.fasta ragtag.scaffold.renamed.fasta
The output assembly obtained by running above script is used as our final assembly ragtag.scaffold.fasta was renamed and changes in the header as CRW_assembly.fasta and ran BUSCO on it before processing by blobtools. Blobtoolsare a modular command-line solution for removing contaminats from associated microorganisms and other non target organisms by better visualisation, quality control and taxonomic partitioning of genome datasets. This will aid in improving our assemblies by screening of the final assemblies produced from ragtag2 for potential contaminants.
Script for BUSCO
#!/bin/bash -e
#SBATCH --nodes 1
#SBATCH --cpus-per-task 1
#SBATCH --ntasks 16
#SBATCH --job-name busco.crw
#SBATCH --mem=30G
#SBATCH --time=72:00:00
#SBATCH --account=uoo02772
#SBATCH --output=%x_%j.out
#SBATCH --error=%x_%j.err
#SBATCH --mail-type=ALL
#SBATCH --mail-user=katma889@student.otago.ac.nz
#SBATCH --hint=nomultithread
module load BUSCO/5.2.2-gimkl-2020a
cp -r $AUGUSTUS_CONFIG_PATH /nesi/nobackup/uoo02772/bin/MyAugustusConfig
export AUGUSTUS_CONFIG_PATH=/nesi/nobackup/uoo02772/bin/MyAugustusConfig
busco --in ../CRW_assembly.fasta --out Busco -c 16 -m genome -l insecta_odb10
we intalled the 'BlobTools2' and its dependencies and then fetch the 'nt database' and 'UniProt database' and formattted it as per the 'Blobtools' requiremnts. For formatting the UniProt databases we downloaded the 'NCBI taxdump' and then uncompressed in a sister directory in a few steps. We also have to fetch the BUSCO lineages that we plan to use such as eukaryota or insecta. For this we followed this. The steps of actions we follwed are given below;
By mapping the ont long reads to our final assembly we got coverage data as bam file.
Script for Coverage
#!/bin/bash -e
#SBATCH --nodes 1
#SBATCH --cpus-per-task 1
#SBATCH --ntasks 20
#SBATCH --job-name coverage.crw
#SBATCH --mem=80G
#SBATCH --time=08:00:00
#SBATCH --account=uoo02772
#SBATCH --output=%x_%j.out
#SBATCH --error=%x_%j.err
#SBATCH --mail-type=ALL
#SBATCH --mail-user=katma889@student.otago.ac.nz
#SBATCH --hint=nomultithread
module load minimap2/2.18-GCC-9.2.0
module load SAMtools/1.12-GCC-9.2.0
minimap2 -ax map-ont -t 20 CRW_assembly.fasta \
crw_ont_nanolyse_porechop_nanofilt.fastq.gz | samtools sort -@10 -O BAM -o CRW_Assembly.bam -
Following the Blobtools manual we blasted our assembly against nt database
Script for blastn
#!/bin/bash -e
#SBATCH --nodes 1
#SBATCH --cpus-per-task 1
#SBATCH --ntasks 36
#SBATCH --job-name bl.crw.xaa
#SBATCH --mem=20G
#SBATCH --time=3-00:00:00
#SBATCH --account=uoo02772
#SBATCH --output=%x_%j.out
#SBATCH --error=%x_%j.err
#SBATCH --mail-type=ALL
#SBATCH --mail-user=katma889@student.otago.ac.nz
#SBATCH --hint=nomultithread
module load BLAST/2.12.0-GCC-9.2.0
export BLASTDB='/path/to/nt'
blastn -db nt \
-query ./CRW_assembly.fasta
-outfmt "6 qseqid staxids bitscore std" \
-max_target_seqs 10 \
-max_hsps 1 \
-evalue 1e-25 \
-num_threads 36 \
-out results/CRW_Assembly.ncbi.blastn.out
As mentioned earlier we downloaded the uniprot database and formatted them following the instrictions here website. Then we blasted our assembly against the uniprot database.
Script for Diamond.blastx
#!/bin/bash -e
#SBATCH --nodes 1
#SBATCH --cpus-per-task 1
#SBATCH --ntasks 10
#SBATCH --job-name dia.xag
#SBATCH --mem=20G
#SBATCH --time=2-00:00:00
#SBATCH --account=uoo02772
#SBATCH --output=%x_%j.out
#SBATCH --error=%x_%j.err
#SBATCH --mail-type=ALL
#SBATCH --mail-user=katma889@student.otago.ac.nz
#SBATCH --hint=nomultithread
module load DIAMOND/2.0.6-GCC-9.2.0
diamond blastx \
--query xag \
--db /path/to/UniProt/reference_proteomes.dmnd \
--outfmt 6 qseqid staxids bitscore qseqid sseqid pident length mismatch gapopen qstart qend sstart send evalue bitscore \
--sensitive \
--max-target-seqs 1 \
--evalue 1e-25 \
--threads 10 \
--memory-limit 100 \
--out xag.blastx.out
Blobdatabase folder was created and then we added the results from blastn, diamond.blastx and coverage information in that folder. We download taxdump dataset as described here and then created a textfile named CRW_assembly.yaml which is included in the script below
assembly:
accession: 000_000000000.0
alias: S_obsoleuts
bioproject: 00000000
biosample: 00000000000
record_type: scaffolds
taxon:
name: Sitona obsoletus
Script used to create, add and filter database
/path/to/blobtools create \
--fasta ../CRW_assembly.fasta \
--meta ../CRW_assembly.yaml \
--taxid 1541163 \
--taxdump path/to/taxdump/ \
CRW_Assembly
/path/to/blobtools add \
--hits ../blastn/blastn.out \
--hits ../diamondx/diamond.blastx.out \
--cov ../coverage/CRW_Assembly.bam \
--taxrule bestsumorder \
--taxdump path/to/taxdump/ \
CRW_Assembly
/path/to/blobtools filter \
--param bestsumorder_superkingdom--Keys=Bacteria \
--param bestsumorder_superkingdom--Keys=Viruses \
--param CRW_Assembly_cov--Min=5 \
--param length--Min=1000 \
--fasta ../CRW_assembly.fasta \
--output ./filtered.bacteria.viruses.minl1000.covmin5 \
CRW_Assembly
Extract the shorter scaffolds and scaffolds with low coverage which were earlier discarded by blobtools filter
/path/to/blobtools/ filter \
--param CRW_Assembly_cov--Min=5 \
--param length--Min=1000 \
--fasta ../CRW_assembly.fasta \
--invert \
--output ./filter.mincov5.minlen1000.invert \
CRW_Assembly
By running above script we got our output as
- Filtered assembly
CRW_Assembly.filtered.fastastored in path/to/output/folder/filter - Inverse filtered file
CRW_Assembly.filtered.inverse.fastastored in path/to/output/folder/filter.inverse
Then we used RagTag to scaffold our assembly CRW_Assembly.filtered.fasta using CRW_Assembly.filtered.inverse.fasta
Script for RagTag
#!/bin/bash -e
#SBATCH --nodes 1
#SBATCH --cpus-per-task 1
#SBATCH --ntasks 10
#SBATCH --job-name ragtag.crw
#SBATCH --mem=6G
#SBATCH --time=04:00:00
#SBATCH --account=uoo02772
#SBATCH --output=%x_%j.out
#SBATCH --error=%x_%j.err
#SBATCH --mail-type=ALL
#SBATCH --mail-user=katma889@student.otago.ac.nz
#SBATCH --hint=nomultithread
export PATH="/nesi/nobackup/uoo02752/nematode/bin/miniconda3/bin:$PATH"
ragtag.py scaffold CRW_assembly.invert.minl1000.covmin5.fasta CRW_assembly.filtered.fasta
Altogether we ran RagTag four times in a series (one after another) as taking the output from earlier ragtag assembly as input for the next one along with CRW_Assembly.filtered.inverse.fasta as input until the fourth run. We also used the oneliner (given below) to rename the sequence header on the ragtag output fasta file.
awk '/^>/{print ">Scaff_" ++i; next}{print}' Ragtag_assembly.fasta > Ragtag_assembly_header_renamed.fasta
This is the final step in our assembly where we used the Illumina short read data to polish our assembly using Pilon. We ran this on two iterations to maximize the quality of the output.
Script for Pilon
#!/bin/bash -e
#SBATCH --nodes 1
#SBATCH --cpus-per-task 1
#SBATCH --ntasks 16
#SBATCH --job-name pilon_crw
#SBATCH --mem=200G
#SBATCH --time=30:00:00
#SBATCH --account=uoo02772
#SBATCH --output=%x_%j.out
#SBATCH --error=%x_%j.err
#SBATCH --mail-type=ALL
#SBATCH --mail-user=katma889@student.otago.ac.nz
#SBATCH --hint=nomultithread
module load BWA/0.7.17-gimkl-2017a
module load SAMtools/1.12-GCC-9.2.0
module load Python/3.9.5-gimkl-2020a
module load Pilon/1.24-Java-15.0.2
#bwa index ragtag.scaffold.fasta
#bwa mem -t 14 ragtag.scaffold.fasta ./NT4_10_8_17_1.trimmed.fastq.gz ./NT4_10_8_17_2.trimmed.fastq.gz | samtools view - -Sb |samtools sort - -@14 -o ./mapping.sorted.bam
#samtools index mapping.sorted.bam
java -Xmx200G -jar $EBROOTPILON/pilon.jar --genome ragtag.scaffold.fasta --fix all --changes --frags mapping.sorted.bam --threads 16 --output CRW_assembly.pilon.1 | tee Pilon.Rnd1.pilon