EasySeq RC-PCR SARS-CoV-2/COVID-19

Variant pipeline V0.4

Table of contents

• GENERAL-INFO
• INSTALL
• RUN
• OUTPUT
• FLOW-DIAGRAM
• TOOLS
• DOCKER
• CONTRIBUTORS
• REMARKS
• REFERENCE
• DISCLAIMER
• LICENSE

General info

This github repository contains an automated pipeline dedicated to properly analyse the EasySeq SARS-CoV-2 (COVID-19) sequence sequencing data. All validation are done using 149 bp or 151 bp paired-end reads.

In short:

• Automated pipeline to analyse Illumina EasySeq COVID-19 samples to a variant report
• The pipeline cleans the Illumina sequencing data
• Uses the SARS-CoV-2 reference genome (NC_045512.2)
• Custom EasySeq Primer filtering and correction
• Mutations and deletions are measured
• Fasta consensus of the sample is created
• Lineage is determined
• Output is available in a structured way
• Full QC reports are created
• PDF and HTML report as output

INSTALL

1. install docker on your OS
2. docker pull jonovox/easyseq_covid19:latest
3. download the newest release of the pipeline via https://github.com/JordyCoolen/easyseq_covid19/releases
4. extract the source code
5. go into the extracted/project folder
6. download conda environments via: https://surfdrive.surf.nl/files/index.php/s/ggoLXzMoa5iSZYa
7. extract conda.tar.gz into the project folder created at step 5
8. Proceed to RUN examples

RUN

RUN_option1

• now you have to perform the test to set everything in place
• first time running the variant pipeline will deploy more conda environments needed to successfully install the pipeline. This can take a while.
• open docker runtime container from image with write rights

sh docker/run.sh covid jonovox/easyseq_covid19:latest

• run the test sample inside the container

nextflow run COVID.nf --sampleName test -resume --outDir /workflow/output/test --reads "/workflow/input/test_OUT01_R{1,2}.fastq.gz"

RUN_option2

• you can also execute multiple samples in non-parallel way

bash scripts/run_batch.sh <path to folders containing the fastq.gz file> <extension of files> jonovox/easyseq_covid19:latest

OUTPUT

/workflow/output/test
|-- HV69-70
|   |-- test.aln
|   |-- test.frag.gz
|   |-- test.fsa
|   |-- test.res
|   `-- test_HVdel.vcf
|-- QC
|   |-- multiqc_data
|   |   |-- multiqc.log
|   |   |-- multiqc_data.json
|   |   |-- multiqc_fastp.txt
|   |   |-- multiqc_general_stats.txt
|   |   |-- multiqc_snpeff.txt
|   |   `-- multiqc_sources.txt
|   |-- multiqc_report.html
|   |-- test.fastp.json
|   |-- test.mosdepth.global.dist.txt
|   |-- test.mosdepth.summary.txt
|   |-- test.per-base.bed.gz
|   |-- test.per-base.bed.gz.csi
|   `-- test_snpEff.csv
|-- annotation
|   |-- snpEff_summary.html
|   |-- test_annot_table.txt
|   |-- test_snpEff.csv
|   `-- test_snpEff.genes.txt
|-- lineage
|   `-- lineage_report.csv
|-- mapping
|   |-- test.bam
|   |-- test.bam.bai
|   |-- test.final.bam
|   `-- test.final.bam.bai
|-- rawvcf
|   `-- test.vcf.gz
|-- report
|   |-- test.fasta
|   |-- test.html
|   `-- test.pdf
|-- uncovered
|   |-- test_noncov.bed
|   `-- test_ubiq.bed
|-- vcf
|   |-- notpassed
|   |   `-- test_3G.txt
|   |-- test.vcf
|   |-- test_final.vcf
|   `-- test_table.txt
`-- vcf_index
    |-- test_concat.vcf.gz
    `-- test_concat.vcf.gz.csi

FLOW-DIAGRAM

TOOLS

• nextflow
• python
• conda/bioconda
• fastp
• BWA MEM
• samtools
• bcftools
• mosdepth
• bedtools
• snpEff
• KMA
• multiQC
• pangolin v2.1.11

DOCKER

build your own docker image

cd easyseq_covid19
docker build --rm -t <image name> ./

CONTRIBUTORS

Department of Medical Microbiology and Radboudumc Center for Infectious Diseases, Radboud university medical center, Nijmegen, The Netherlands

• J.P.M. Coolen (jordy.coolen@radboudumc.nl)

NimaGen B.V., Nijmegen, The Netherlands

• R.A. Lammerts (NimaGen B.V., Nijmegen, The Netherlands)
• J.T. Vonk (Student HAN Bioinformatics, Nijmegen, The Netherlands)

REMARKS

spike S
21765-21770HV 69-70 deletion

The current EasySeq design is not completly overlapping the region 21765-21770 / HV 69-70.

---->         To solve this a template based strategy using KMA is applied.
      <---    This method measures which template matches best. Either Wildtype (NC_045512.2) or
              a variant containing the 21765-21770 / HV 69-70 deletion. The result of this strategy
              is projected in the VCF to ensure correct output. This works perfect for now because no other deletions are
              known on this exact location.

REFERENCE

For citing this work please cite:

Novel SARS-CoV-2 Whole-genome sequencing technique using Reverse Complement PCR enables easy, fast and accurate outbreak analysis in hospital and community settings Femke Wolters, Jordy P.M. Coolen, Alma Tostmann, Lenneke F.J. van Groningen, Chantal P. Bleeker-Rovers, Edward C.T.H. Tan, Nannet van der Geest-Blankert, Jeannine L.A. Hautvast, Joost Hopman, Heiman F.L. Wertheim, Janette C. Rahamat-Langendoen, Marko Storch, Willem J.G. Melchers bioRxiv 2020.10.29.360578; doi: https://doi.org/10.1101/2020.10.29.360578.

Also cite the other programs used, see list of used tools

The work is currently under revision.

DISCLAIMER

This is for Research Only. The code and pipeline is continuously under development. We cannot guarantee a full error free result. Especially with the fast developments in SARS-CoV-2/COVID-19 sequencing and the continuously mutating nature of the virus.

LICENSE