wf-flu | Influenza Typing Workflow
This repository contains a nextflow workflow that takes targeted ONT Influenza sequencing data to produce typing information.
Introduction
Influenza is a single-stranded RNA virus and contains a 13.5-14.5kb genome which is split into 8 segments encoding 10-14 proteins (dependent on strain).
The virus is classified using two proteins found on the outer surface of the viral capsid. You’ve probably heard of H1N1 Influenza for example. The H represents hemagglutinin and the N is neuraminidase.
The Oxford Nanopore Technologies protocol listed here amplifies segments of the Influenza Type A and Type B genomes. Using this analysis workflow, users can determine the most likely strain of Influenza to which the sample being sequenced belongs.
Data Analysis
Workflow steps:
- Concatenate reads & filter out short reads < 200 bases long
- Align reads to reference (minimap2)
- Coverage calculations (samtools)
- Call variants with medaka
- Make a (coverage masked) consensus with bcftools
- Type with abricate
Downsampling
Downsampling is optional
For every segment in the reference genome:
- Get the length
- Work out +/- 10%
- Filter reads within that segment and within +/- 10% of segment length
Typing
Typing is carried out using abricate using the insaflu database containing the following sequences:
| Database | Gene | Accession | Details |
|---|---|---|---|
| insaflu | M1 | MK576795 | Type_A MK576795 A/England/7821/2019 2019/01/03 7 (MP) |
| insaflu | M1 | AF100378 | Type_B AF100378.1 Influenza B virus B/Yamagata/16/88 segment 7 M1 matrix protein (M) and BM2 protein (BM2) genes, complete cds |
| insaflu | HA | FJ966974 | H1 FJ966974.1 Influenza A virus (A/California/07/2009(H1N1)) segment 4 hemagglutinin (HA) gene, complete cds |
| insaflu | HA | L11142 | H2 L11142.1 Influenza A virus (A/Singapore/1/57 (H2N2)) hemagglutinin (HA) gene, complete cds |
| insaflu | HA | MK576794 | H3 MK576794 A/England/7821/2019 2019/01/03 4 (HA) |
| insaflu | HA | AF285883 | H4 AF285883.2 Influenza A virus (A/Swine/Ontario/01911-2/99 (H4N6)) segment 4 hemagglutinin (HA) gene, complete cds |
| insaflu | HA | EF541403 | H5 EF541403.1 Influenza A virus (A/Viet Nam/1203/2004(H5N1)) segment 4 hemagglutinin (HA) gene, complete cds |
| insaflu | HA | AB295613 | H15 AB295613.1 Influenza A virus (A/duck/Australia/341/83(H15N8)) HA gene for haemagglutinin, complete cds |
| insaflu | NA | GQ377078 | N1 GQ377078.1 Influenza A virus (A/California/07/2009(H1N1)) segment 6 neuraminidase (NA) gene, complete cds |
| insaflu | NA | MK576796 | N2 MK576796 A/England/7821/2019 2019/01/03 6 (NA) |
| insaflu | NA | AB295614 | N8 AB295614.1 Influenza A virus (A/duck/Australia/341/83(H15N8)) NA gene for neuraminidase, complete cds |
| insaflu | HA | AY338459 | H7 AY338459.1 Influenza A virus (A/Netherlands/219/2003(H7N7)) segment 4 hemagglutinin (HA) gene, complete cds |
| insaflu | HA | CY014659 | H8 CY014659.1 Influenza A virus (A/turkey/Ontario/6118/1968(H8N4)) segment 4, complete sequence |
| insaflu | HA | CY014694 | H13 CY014694.1 Influenza A virus (A/gull/Maryland/704/1977(H13N6)) segment 4, complete sequence |
| insaflu | HA | CY018765 | Yamagata CY018765.1 Influenza B virus (B/Yamagata/16/1988) segment 4, complete sequence |
| insaflu | HA | CY103892 | H17 CY103892.1 Influenza A virus (A/little yellow-shouldered bat/Guatemala/060/2010(H17N10)) hemagglutinin (HA) gene, complete cds |
| insaflu | NA | CY103894 | N10 CY103894.1 Influenza A virus (A/little yellow-shouldered bat/Guatemala/060/2010(H17N10)) neuraminidase (NA) gene, complete cds |
| insaflu | NA | CY125730 | N3v2 CY125730.1 Influenza A virus (A/Mexico/InDRE7218/2012(H7N3)) neuraminidase (NA) gene, complete cds |
| insaflu | HA | CY125945 | H18 CY125945.1 Influenza A virus (A/flat-faced bat/Peru/033/2010(H18N11)) hemagglutinin (HA) gene, complete cds |
| insaflu | NA | CY125947 | N11 CY125947.1 Influenza A virus (A/flat-faced bat/Peru/033/2010(H18N11)) neuraminidase-like protein (NA) gene, complete cds |
| insaflu | HA | CY130078 | H12 CY130078.1 Influenza A virus (A/duck/Alberta/60/1976(H12N5)) hemagglutinin (HA) gene, complete cds |
| insaflu | HA | CY130094 | H14 CY130094.1 Influenza A virus (A/mallard/Astrakhan/263/1982(H14N5)) hemagglutinin (HA) gene, complete cds |
| insaflu | NA | CY130096 | N5 CY130096.1 Influenza A virus (A/mallard/Astrakhan/263/1982(H14N5)) neuraminidase (NA) gene, complete cds |
| insaflu | HA | DQ376624 | H6 DQ376624.1 Influenza A virus (A/chicken/Taiwan/0705/99(H6N1)) hemagglutinin (HA) gene, complete cds |
| insaflu | HA | EU293864 | H16 EU293864.1 Influenza A virus (A/black-headed gull/Turkmenistan/13/76(H16N3)) hemagglutinin (HA) gene, complete cds |
| insaflu | HA | FJ183474 | H10 FJ183474.1 Influenza A virus (A/mallard/Bavaria/3/2006(H10N7)) segment 4 hemagglutinin (HA) gene, complete cds |
| insaflu | NA | FJ183475 | N7 FJ183475.1 Influenza A virus (A/mallard/Bavaria/3/2006(H10N7)) segment 6 neuraminidase (NA) gene, complete cds |
| insaflu | NA | GQ907296 | N3v1 GQ907296.1 Influenza A virus (A/black headed gull/Mongolia/1756/2006(H16N3)) segment 6 neuraminidase (NA) gene, complete cds |
| insaflu | HA | GU052203 | H11 GU052203.1 Influenza A virus (A/duck/England/1/1956(H11N6)) segment 4 hemagglutinin (HA) gene, complete cds |
| insaflu | NA | KC853765 | N9 KC853765.1 Influenza A virus (A/Hangzhou/1/2013(H7N9)) segment 6 neuraminidase (NA) gene, complete cds |
| insaflu | HA | KX879589 | H9 KX879589.1 Influenza A virus (A/swine/Hong Kong/9/98(H9N2)) segment 4 hemagglutinin (HA) gene, partial cds |
| insaflu | HA | M58428 | Victoria M58428.1 Influenza B/Victoria/2/87, hemagglutinin (seg 4), RNA |
| insaflu | NA | EU429793 | N4 EU429793.1 Influenza A virus (A/turkey/Ontario/6118/1968(H8N4)) segment 6 neuraminidase (NA) mRNA, complete cds |
| insaflu | NA | EU429795 | N6 EU429795.1 Influenza A virus (A/duck/England/1/1956(H11N6)) segment 6 neuraminidase (NA) mRNA, complete cds |
Quickstart
The workflow uses nextflow to manage compute and software resources. Thus, nextflow will need to be installed before attempting to run the workflow.
The workflow can currently be run using either Docker or Singularity to provide isolation of the required software. Both methods are automated out-of-the-box, provided either docker of singularity is installed.
It is not required to clone or download the git repository in order to run the workflow. For more information on running EPI2ME Labs workflows visit our website.
Workflow options
To obtain the workflow, having installed nextflow, users can run:
nextflow run epi2me-labs/wf-flu --help
to see the options for the workflow.
Workflow outputs
The workflow creates several files that are useful for interpretation and analysis:
- Per run:
wf-flu-report.html: Easy-to-use HTML report for all samples in the runwf-flu-results.csv: Typing results in CSV format for onward processing
- Per sample:
<SAMPLE_NAME>.stats: Read stats<SAMPLE_NAME>.bam: Alignment of reads to reference<SAMPLE_NAME>.bam.bai: BAM index<SAMPLE_NAME>.annotate.filtered.vcf: medaka called variants<SAMPLE_NAME>.draft.consensus.fasta: Consensus FASTA<SAMPLE_NAME>.insaflu.typing.txt: abricate typing results