Sep 02, 2022

Public workspaceDe-novo assembly of Xanthomonas genomes from Illumina NovaSeq reads

DOI
https://dx.doi.org/10.17504/protocols.io.kxygxzrqzv8j/v1
  • David J Studholme1,
  • Jamie Harrison1
  • 1University of Exeter
David J Studholme
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DOI: https://dx.doi.org/10.17504/protocols.io.kxygxzrqzv8j/v1
Protocol Citation: David J Studholme, Jamie Harrison 2022. De-novo assembly of Xanthomonas genomes from Illumina NovaSeq reads. protocols.io https://dx.doi.org/10.17504/protocols.io.kxygxzrqzv8j/v1
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: August 19, 2022
Last Modified: September 02, 2022
Protocol Integer ID: 68908
Keywords: xanthomonas genome sequence, novo assembly of xanthomona, xanthomona, sequencing data, raw sequence read, quality control of the raw sequence read, read genomic shotgun, novo assembly, alignment of read, illumina novaseq, preliminary assembly, assembly
Funders Acknowledgements:
UKRI
Grant ID: BB/T010916/1
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Abstract
This protocol describes the de-novo assembly of Xanthomonas genome sequences from short-read genomic shotgun sequencing data. It includes quality control of the raw sequence reads, assembly and finally polishing of the assembly based on alignment of reads against the preliminary assembly.
Troubleshooting
Software pre-requisites.
Note
This protocol assumes that you have already installed fastp, SPAdes, SAMtools, BowTie2 and Pilon. I also assumes that the paired Illumina sequence data comprises two gzipped FASTQ files called name_r1.fq.gz and name_r1.fq.gz.

Perform quality-based filtering and adapter trimming using fastp.

Command
Creates a directory for the fastp QC report files.
new command name
mkdir name_fastp_out

Command
Generates trimmed and filtered sequence files and QC reports on the Illumina NovaSeq FASTQ sequence files.
new command name
fastp -i name_r1.fq.gz -I name_r2.fq.gz -o name_trimmed_r1.fq.gz -O name_trimmed_r2.fq.gz --unpaired1 name_trimmed_unp.fq.gz --unpaired2 name_trimmed_unp.fq.gz -r --cut_right_window_size 5 --cut_right_mean_quality 20 -c -l 50 -j name_fastp_out/name_fastp_report.json -h name_fastp_out/name_fastp_report.html

Perform de-novo assembly using SPAdes.


Command
Performs the de-novo assembly.
new command name
spades.py -1 name_trimmed_r1.fq.gz -2 name_trimmed_r2.fq.gz -s name_trimmed_unp.fq.gz --careful --cov-cutoff auto -o name_spades_out

Polishing with Pilon

Note
This step assumes that the SPAdes assembly is contained in a file in the current working directory called name.fasta. It is assumed that the two trimmed-and-filtered gzipped FASTQ files are also in the current working directory. If these files are located elsewhere, then you can make symbolic links to them in the current working directory.

Command
Creates BowTie2 index files with 'name' as the prefix for their filenames.
new command name
bowtie2-build name.fasta name

Command
Performs alignment of the trimmed-and-filtered reads against the genome assembly to generate an alignment in SAM format.
new command name
bowtie2 -x name -1 name_trimmed_r1.fq.gz -2 name_trimmed_r2.fq.gz -S name_vs_name.sam

Command
Converts the SAM-formatted file into BAM format.
new command name
samtools view -b -T name.fasta name_vs_name.sam -o name_vs_name.sam.bam

Command
Sorts the BAM file.
new command name
samtools sort --reference name.fasta name_vs_name.sam.bam -o name_vs_name.sam.bam.sorted.bam

Command
Indexes the sorted BAM file.
new command name
samtools index name_vs_name.sam.bam.sorted.bam

Command
Removes the intermediate files to save disk space.
new command name
rm name_vs_name.sam.bam $name_vs_$name.sam

Command
Generates a modified genome assembly based on reconciling discrepancies between assembly and aligned reads.
new command name
pilon --genome name.fasta --frags name_vs_name.sam.bam.sorted.bam --output name.pilon --outdir name_pilon_out



Expected result
The polished genome assembly in FASTA format can be found in the Pilon output directory: ./name_pilon_out/name.pilon.fasta

This file can now be subjected to further quality control and/or submitted to public repositories.



Bibliography


Citation
Chen S, Zhou Y, Chen Y, Gu J (2018). fastp: an ultra-fast all-in-one FASTQ preprocessor. Bioinformatics (Oxford, England).
https://doi.org/10.1093/bioinformatics/bty560
LINK

Citation
Bankevich A, Nurk S, Antipov D, Gurevich AA, Dvorkin M, Kulikov AS, Lesin VM, Nikolenko SI, Pham S, Prjibelski AD, Pyshkin AV, Sirotkin AV, Vyahhi N, Tesler G, Alekseyev MA, Pevzner PA (2012). SPAdes: a new genome assembly algorithm and its applications to single-cell sequencing. Journal of computational biology : a journal of computational molecular cell biology.
https://doi.org/10.1089/cmb.2012.0021
LINK

Citation
Danecek P, Bonfield JK, Liddle J, Marshall J, Ohan V, Pollard MO, Whitwham A, Keane T, McCarthy SA, Davies RM, Li H (2021). Twelve years of SAMtools and BCFtools. GigaScience.
https://doi.org/pii:giab008.10.1093/gigascience/giab008
LINK

Citation
Langmead B, Salzberg SL (2012). Fast gapped-read alignment with Bowtie 2. Nature methods.
https://doi.org/10.1038/nmeth.1923
LINK

Citation
Walker BJ, Abeel T, Shea T, Priest M, Abouelliel A, Sakthikumar S, Cuomo CA, Zeng Q, Wortman J, Young SK, Earl AM (2014). Pilon: an integrated tool for comprehensive microbial variant detection and genome assembly improvement. PloS one.
https://doi.org/10.1371/journal.pone.0112963
LINK

Citations
Step 5
Chen S, Zhou Y, Chen Y, Gu J. fastp: an ultra-fast all-in-one FASTQ preprocessor.
https://doi.org/10.1093/bioinformatics/bty560
Step 5
Bankevich A, Nurk S, Antipov D, Gurevich AA, Dvorkin M, Kulikov AS, Lesin VM, Nikolenko SI, Pham S, Prjibelski AD, Pyshkin AV, Sirotkin AV, Vyahhi N, Tesler G, Alekseyev MA, Pevzner PA. SPAdes: a new genome assembly algorithm and its applications to single-cell sequencing.
https://doi.org/10.1089/cmb.2012.0021
Step 5
Danecek P, Bonfield JK, Liddle J, Marshall J, Ohan V, Pollard MO, Whitwham A, Keane T, McCarthy SA, Davies RM, Li H. Twelve years of SAMtools and BCFtools.
https://doi.org/pii:giab008.10.1093/gigascience/giab008
Step 5
Langmead B, Salzberg SL. Fast gapped-read alignment with Bowtie 2.
https://doi.org/10.1038/nmeth.1923
Step 5
Walker BJ, Abeel T, Shea T, Priest M, Abouelliel A, Sakthikumar S, Cuomo CA, Zeng Q, Wortman J, Young SK, Earl AM. Pilon: an integrated tool for comprehensive microbial variant detection and genome assembly improvement.
https://doi.org/10.1371/journal.pone.0112963