Jun 12, 2024
Version 2
Genome assembly (Nanopore and Illumina reads) V.2
- 1Universidade Federal do Sul da Bahia
Protocol Citation: Rafael Rodrigues Ferrari, Thiago Mafra Batista 2024. Genome assembly (Nanopore and Illumina reads). protocols.io https://dx.doi.org/10.17504/protocols.io.kxygxywyol8j/v2Version created by Thiago Mafra Batista
Manuscript citation:
Ferrari RR, Batista TM, Zhou QS, Hilário HO, Orr MC, Luo A, Zhu CD. The Whole Genome of Colletes collaris (Hymenoptera: Colletidae): An Important Step in Comparative Genomics of Cellophane Bees. Genome Biol Evol. 2023 May 5;15(5):evad062. doi: 10.1093/gbe/evad062. PMID: 37075227; PMCID: PMC10159585.
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: June 11, 2024
Last Modified: June 12, 2024
Protocol Integer ID: 101683
Keywords: long reads, nanopore reads, genome assembly, genome polishment, bioinformatics
Funders Acknowledgement:
Suzano Papel e Celulose S/A
Grant ID: 23746.009802/2021-88
Veracel Celulose
Grant ID: 23746.001092/2022-30
Abstract
This protocol offers detailed, step-by-step instructions for students and researchers to assemble nuclear genomes using long reads generated by Nanopore technology. Before assembling the genome, we will align the reads against a bacterial genome database to eliminate potential contamination. The assembled contigs will then be polished using Illumina short reads.
SEQUENCING QUALITY CHECK
SEQUENCING QUALITY CHECK
****LongQC (https://github.com/yfukasawa/LongQC)****
***Prepare a .pbs file to run the analysis remotely on Sagarana***
python /home/fafinha/bin/LongQC/longQC.py sampleqc -x ont-ligation -c /tmp/LongQC_run/reads_trim.fq \
-p 64 -o /tmp/LongQC_run /home/fafinha/colletes_collaris/reads/genomic_reads/longreads_rawdata_collaris.fq
mv /tmp/LongQC_run/ /home/fafinha/colletes_collaris/
CROSS-SPECIES CONTAMINATION FILTERIN
CROSS-SPECIES CONTAMINATION FILTERIN
****Magic-BLAST (https://ncbi.github.io/magicblast/)****
***Index the database***
$~/bin/ncbi-magicblast-1.7.0/bin/makeblastdb -in refseq_release_215_bacteria.fna -dbtype nucl
***ONT whole-genome sequencing***
**Prepare a .pbs file to run the analysis remotely on Sagarana**
magicblast -db /databases/ref_prok_rep_genomes_out20/ref_prok_rep_genomes \
-query /home/fafinha/collaris/reads/genomic_reads/reads/genomic_reads/ONT_longreads_rawdata_collaris.fq \
-out_unaligned ONT_longreads_unaligned_in_refseq_prok_collaris.fa -num_threads 80 -infmt fastq -unaligned_fmt fasta > output.sam
***Illumina whole-genome sequencing***
**Prepare a .pbs file to run the analysis remotely on Sagarana**
magicblast -db /databases/ref_prok_rep_genomes_out20/ref_prok_rep_genomes -query /home/fafinha/collaris/reads/genomic_reads/Illumina_shortreads.R1.fastq \
-query_mate /home/fafinha/collaris/reads/genomic_reads/Illumina_shortreads.R2.fastq \
-paired -no_discordant -infmt fastq -unaligned_fmt sam -num_threads 128 \
-out_unaligned /home/fafinha/collaris/mafra/descontamination/illumina_reads/illumina_unaligned_in_refseq_prok.sam \
-out /home/fafinha/collaris/mafra/descontamination/illumina_reads/illumina_aligned_in_refseq_prok.sam
***Convert output file***
$/programs/samtools-1.12/bin/samtools view -Sb -@12 illumina_unaligned_in_refseq_prok.sam > illumina_unaligned_in_refseq_prok.bam
$/programs/samtools-1.12/bin/samtools sort illumina_unaligned_in_refseq_prok.bam -o illumina_unaligned_in_refseq_prok_sorted.bam -@12
$/programs/samtools-1.12/bin/samtools fastq -1 paired1.fq -2 paired2.fq -n illumina_unaligned_in_refseq_prok_sorted.bam -@12
GENOME SIZE ESTIMATION
GENOME SIZE ESTIMATION
****Jellyfish (https://github.com/gmarcais/Jellyfish)****
***Counting k-mers***
**Prepare a .pbs file to run the analysis remotely on Sagarana**
/programs/jellyfish/jellyfish-2.3.0 count -C -m 21 -s 10G -t 36 /home/fafinha/collaris/reads/genomic_reads/D2015099C_L4_304X04.R1.fastq \ /home/fafinha/collaris/reads/genomic_reads/D2015099C_L4_304X04.R2.fastq -o /home/fafinha/collaris/Jellyfish/reads.jf
/programs/jellyfish/jellyfish-2.3.0 histo -t 36 /home/fafinha/collaris/Jellyfish/reads.jf > /home/fafinha/collaris/Jellyfish/reads.histo
**Size estimation**
/////STRATEGY #1: GenomeScope (on my PC)\\\\\
*Go to the directory where reads.histo is located*
$/home/rafael/genomescope2.0/genomescope.R -i reads.histo -o output -k 21
/////STRATEGY #2: R (on my PC)\\\\\
*Go to the directory where reads.histo is located*
$R
$library ("findGSE")
$findGSE(histo="reads.histo", sizek=21, outdir="21mer")
GENOME ASSEMBLY
GENOME ASSEMBLY
****NextDenovo (https://github.com/Nextomics/NextDenovo)****
***Prepare an 'input.fofn' file***
$ls /home/fafinha/collaris/reads/genomic_reads/ONT_longreads_rawdata_collaris.fq > input.fofn
***Prepare a 'run.cfg' file***
[General]
job_type = local
job_prefix = nextDenovo
task = all
rewrite = yes
deltmp = yes
parallel_jobs = 24
input_type = raw
read_type = ont
input_fofn = /home/fafinha/collaris/NextDenovo_run/input.fofn
workdir = 01_rundir
[correct_option]
read_cutoff = 1k
genome_size = 300m
sort_options = -m 20g -t 8
minimap2_options_raw = -t 8
pa_correction = 3
correction_options = -p 15
[assemble_option]
minimap2_options_cns = -t 8
nextgraph_options = -a 1
***Prepare a .pbs file to run the analysis remotely on Sagarana***
/home/fafinha/bin/NextDenovo/nextDenovo /home/fafinha/collaris/NextDenovo_run/run.cfg
GENOME ASSEMBLY STATISTICS
****scaffolds_stats****
***Compare two runs and include the stats into a .txt***
$scaffold_stats.pl -f run1/assembly.fasta run2/assembly.fasta -N 1 -t 1000 10000 | tee stats.txt
#BBMap is part of BBTools (https://jgi.doe.gov/data-and-tools/bbtools/bb-tools-user-guide/)
***Run the script stats.sh using the main output produced by Flye (on my PC)***
$bash /mnt/d/Genomics/bbmap/stats.sh in=nd.asm.fasta out=nd.asm.fasta_stats.txt
****BUSCO (https://busco.ezlab.org/)****
**Run BUSCO (using a docker)**
$docker run --rm -e USERID=$UID -u $UID -v /home/rferrari/:/home/rferrari/ -w /home/rferrari/projetos/collaris/BUSCO_run/genome/post_polishment/NextDenovo/run4_RF_final/SRs ezlabgva/busco:v5.2.2_cv1 busco -i /home/rferrari/projetos/collaris/BUSCO_run/genome/post_polishment/NextDenovo/run4_RF_final/SRs/genome.nextpolish.fasta -l hymenoptera_odb10 --augustus_species Apis_mellifera -o run1 -m geno -c 12
#To see list of available reference datasets
$docker run --rm -e USERID=$UID -u $UID -v /home/rferrari/:/home/rferrari/ -w /home/rferrari ezlabgva/busco:v5.2.2_cv1 busco --list-datasets
GENOME POLISHMENT
GENOME POLISHMENT
****NextPolish (https://github.com/Nextomics/NextPolish)****
***Using only short reads***
**Prepare a 'sgs.fofn' file**
$ls /home/fafinha/collaris/reads/genomic_reads/Illumina_shortreads.R1.fastq /home/fafinha/collaris/reads/genomic_reads/Illumina_shortreads.R2.fastq > sgs.fofn
**Create a 'run.cfg' file**
[General]
job_type = local
job_prefix = nextPolish
task = best
rewrite = yes
rerun = 3
parallel_jobs = 6
multithread_jobs = 5
genome = /home/fafinha/collaris/mafra/flye_run/run1/assembly.fasta
genome_size = auto
workdir = /home/fafinha/collaris/Nextpolish_run/01_rundir
polish_options = -p {multithread_jobs}
[sgs_option]
sgs_fofn = /home/fafinha/collaris/Nextpolish_run/sgs.fofn
sgs_options = -max_depth 100 -bwa
**Prepare a .pbs file to run the analysis remotely on Sagarana**
/programs/NextPolish_n005/nextPolish /home/fafinha/collaris/Nextpolish_run/run.cfg
***Using both short and long reads***
**Prepare a 'sgs.fofn' file**
$ls /home/fafinha/collaris/reads/genomic_reads/Illumina_shortreads.R1.fastq /home/fafinha/collaris/reads/genomic_reads/Illumina_shortreads.R2.fastq > sgs.fofn
**Prepare a 'lgs.fofn' file**
$ls /home/fafinha/collaris/reads/genomic_reads/ONT_longreads_rawdata_collaris.fq > lgs.fofn
**Create a 'run.cfg' file**
[General]
job_type = local
job_prefix = nextPolish
task = best
rewrite = yes
rerun = 3
parallel_jobs = 8
multithread_jobs = 8
genome = /home/fafinha/collaris/mafra/flye_run/run1/assembly.fasta
genome_size = 300m
workdir = /home/fafinha/collaris/NextPolish_run/run5/long_short_reads/01_rundir
polish_options = -p {multithread_jobs}
[sgs_option]
sgs_fofn = /home/fafinha/collaris/NextPolish_run/run5/short_reads/sgs.fofn
sgs_options = -max_depth 100 -bwa
[lgs_option]
lgs_fofn = /home/fafinha/collaris/NextPolish_run/run5/long_short_reads/lgs.fofn
lgs_options = -min_read_len 1k -max_depth 100
lgs_minimap2_options = -x map-ont
**Prepare a .pbs file to run the analysis remotely on Sagarana**
/programs/NextPolish_n005/nextPolish /home/fafinha/collaris/NextPolish_run/run5/long_short_reads/run.cfg