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Influenza Workflow

By Matt Parker
Published in How Tos
August 24, 2022
2 min read
Influenza Workflow

We are pleased to offer a new workflow for the analysis of targeted Oxford Nanopore Technologies sequencing of Influenza virus.

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 the analysis workflow described here users can determine the most likely strain of Influenza to which the sample being sequenced belongs.

As with all our workflows we welcome your comments and suggestions for improvements or any reports of isssues on the nanopore community or on GitHub.

How To Run

You have two options for local analysis of your Influenza data.

1. Command Line

nextflow nextflow run epi2me-labs/wf-flu --fastq <PATH_TO_DEMULTIPLEXED_FASTQS>

Optional parameters include

  • --sample_sheet <PATH_TO_SAMPLESHEET> (Example below)
  • --downsample <NUMBER_OF_READS> (default: None, suggested: 500)
  • --min_qscore <MINIMUM_READ_QSCORE> (default: 9)

Sample Sheet

A sample sheet allows you to name your samples, it must be a comma or tab separated file like below (note: this is equivalent to that which can be provided to MinKNOW, the workflow makes use of only a subset of the columns as shown below):

barcode,sample_id,type
barcode02,H1N1_strain_A-PR-8-34,test_sample
barcode03,H1N1_strain_A-Virginia-ATCC1-2009,test_sample
barcode04,H3N2_strain_A-Virginia-ATCC6-2012,test_sample
barcode08,fluB-BY-massachusetts-2-2012,test_sample
barcode09,fluB_B-taiwan-2-62,test_sample
barcode10,fluB-lee-40,test_sample
barcode31,fluB-yamagata-florida-4-2006_1,test_sample
barcode91,fluB-yamagata-florida-4-2006_2,test_sample
barcode55,fluA_H3N2_A-wisconsin-15-2009,test_sample

2. EPI2ME Labs

You can also use EPI2ME Labs, our straightforward application for the point and click execution of Nextflow workflows. This is available for Windows, MacOS and ubuntu on our downloads page.

EPI2ME Labs Application
EPI2ME Labs Application
EPI2ME Labs Influenza Workflow Settings
EPI2ME Labs Influenza Workflow Settings

Data Analysis Details

The workflow analyses all samples on a multiplexed Influenza sequencing run and provide an easy to interpret report.

The workflow carries out the following steps:

  1. Concatenate reads from the same sample & filter out short reads < 200 bases long
  2. Filter reads below median qscore of 9 (can be adjusted by changing --min_qscore parameter)
  3. Align reads to reference (minimap2)
  4. Optional downsampleing to speed up execution (activate by using --downsample 500)
  5. Coverage calculations (samtools)
  6. Call variants with medaka
  7. Make a (coverage masked) consensus with bcftools
  8. Perform Influenza typing with abricate

Reference

We align to the CDC’s multi-fasta Influenza reference which contains FluA + FluB segments, as well as including alternative segment sequences from disparate strains:

A_MP, A_NP, A_NS, A_PA, A_PB1, A_PB2, A_HA_H1, A_HA_H10, A_HA_H11, A_HA_H12, A_HA_H13, A_HA_H14, A_HA_H15, A_HA_H16, A_HA_H2, A_HA_H3, A_HA_H4, A_HA_H5, A_HA_H6, A_HA_H7, A_HA_H8, A_HA_H9, A_NA_N1, A_NA_N2, A_NA_N3, A_NA_N4, A_NA_N5, A_NA_N6, A_NA_N7, A_NA_N8, A_NA_N9, B_HA, B_MP, B_NA, B_NP, B_NS, B_PA, B_PB1, B_PB2

Downsampling

Downsampling is optional and will speed up workflow execution.

For every segment in the reference genome, the workflow:

  1. Finds the length of the segment
  2. Finds reads within ±10% of the segment length
  3. Collates a balanced set of reads from forward and reverse strands to achieve the desired read count

Typing

Typing is carried out using abricate with the INSaFLU database containing the sequences in the table below.

DatabaseGeneAccessionDetails
insafluM1MK576795Type_A MK576795 A/England/7821/2019 2019/01/03 7 (MP)
insafluM1AF100378Type_B AF100378.1 Influenza B virus B/Yamagata/16/88 segment 7 M1 matrix protein (M) and BM2 protein (BM2) genes, complete cds
insafluHAFJ966974H1 FJ966974.1 Influenza A virus (A/California/07/2009(H1N1)) segment 4 hemagglutinin (HA) gene, complete cds
insafluHAL11142H2 L11142.1 Influenza A virus (A/Singapore/1/57 (H2N2)) hemagglutinin (HA) gene, complete cds
insafluHAMK576794H3 MK576794 A/England/7821/2019 2019/01/03 4 (HA)
insafluHAAF285883H4 AF285883.2 Influenza A virus (A/Swine/Ontario/01911-2/99 (H4N6)) segment 4 hemagglutinin (HA) gene, complete cds
insafluHAEF541403H5 EF541403.1 Influenza A virus (A/Viet Nam/1203/2004(H5N1)) segment 4 hemagglutinin (HA) gene, complete cds
insafluHAAB295613H15 AB295613.1 Influenza A virus (A/duck/Australia/341/83(H15N8)) HA gene for haemagglutinin, complete cds
insafluNAGQ377078N1 GQ377078.1 Influenza A virus (A/California/07/2009(H1N1)) segment 6 neuraminidase (NA) gene, complete cds
insafluNAMK576796N2 MK576796 A/England/7821/2019 2019/01/03 6 (NA)
insafluNAAB295614N8 AB295614.1 Influenza A virus (A/duck/Australia/341/83(H15N8)) NA gene for neuraminidase, complete cds
insafluHAAY338459H7 AY338459.1 Influenza A virus (A/Netherlands/219/2003(H7N7)) segment 4 hemagglutinin (HA) gene, complete cds
insafluHACY014659H8 CY014659.1 Influenza A virus (A/turkey/Ontario/6118/1968(H8N4)) segment 4, complete sequence
insafluHACY014694H13 CY014694.1 Influenza A virus (A/gull/Maryland/704/1977(H13N6)) segment 4, complete sequence
insafluHACY018765Yamagata CY018765.1 Influenza B virus (B/Yamagata/16/1988) segment 4, complete sequence
insafluHACY103892H17 CY103892.1 Influenza A virus (A/little yellow-shouldered bat/Guatemala/060/2010(H17N10)) hemagglutinin (HA) gene, complete cds
insafluNACY103894N10 CY103894.1 Influenza A virus (A/little yellow-shouldered bat/Guatemala/060/2010(H17N10)) neuraminidase (NA) gene, complete cds
insafluNACY125730N3v2 CY125730.1 Influenza A virus (A/Mexico/InDRE7218/2012(H7N3)) neuraminidase (NA) gene, complete cds
insafluHACY125945H18 CY125945.1 Influenza A virus (A/flat-faced bat/Peru/033/2010(H18N11)) hemagglutinin (HA) gene, complete cds
insafluNACY125947N11 CY125947.1 Influenza A virus (A/flat-faced bat/Peru/033/2010(H18N11)) neuraminidase-like protein (NA) gene, complete cds
insafluHACY130078H12 CY130078.1 Influenza A virus (A/duck/Alberta/60/1976(H12N5)) hemagglutinin (HA) gene, complete cds
insafluHACY130094H14 CY130094.1 Influenza A virus (A/mallard/Astrakhan/263/1982(H14N5)) hemagglutinin (HA) gene, complete cds
insafluNACY130096N5 CY130096.1 Influenza A virus (A/mallard/Astrakhan/263/1982(H14N5)) neuraminidase (NA) gene, complete cds
insafluHADQ376624H6 DQ376624.1 Influenza A virus (A/chicken/Taiwan/0705/99(H6N1)) hemagglutinin (HA) gene, complete cds
insafluHAEU293864H16 EU293864.1 Influenza A virus (A/black-headed gull/Turkmenistan/13/76(H16N3)) hemagglutinin (HA) gene, complete cds
insafluHAFJ183474H10 FJ183474.1 Influenza A virus (A/mallard/Bavaria/3/2006(H10N7)) segment 4 hemagglutinin (HA) gene, complete cds
insafluNAFJ183475N7 FJ183475.1 Influenza A virus (A/mallard/Bavaria/3/2006(H10N7)) segment 6 neuraminidase (NA) gene, complete cds
insafluNAGQ907296N3v1 GQ907296.1 Influenza A virus (A/black headed gull/Mongolia/1756/2006(H16N3)) segment 6 neuraminidase (NA) gene, complete cds
insafluHAGU052203H11 GU052203.1 Influenza A virus (A/duck/England/1/1956(H11N6)) segment 4 hemagglutinin (HA) gene, complete cds
insafluNAKC853765N9 KC853765.1 Influenza A virus (A/Hangzhou/1/2013(H7N9)) segment 6 neuraminidase (NA) gene, complete cds
insafluHAKX879589H9 KX879589.1 Influenza A virus (A/swine/Hong Kong/9/98(H9N2)) segment 4 hemagglutinin (HA) gene, partial cds
insafluHAM58428Victoria M58428.1 Influenza B/Victoria/2/87, hemagglutinin (seg 4), RNA
insafluNAEU429793N4 EU429793.1 Influenza A virus (A/turkey/Ontario/6118/1968(H8N4)) segment 6 neuraminidase (NA) mRNA, complete cds
insafluNAEU429795N6 EU429795.1 Influenza A virus (A/duck/England/1/1956(H11N6)) segment 6 neuraminidase (NA) mRNA, complete cds

Output Files

The workflow outputs several files that are useful for interpretation and analysis:

  • Per run:
    • wf-flu-report.html: Easy to use HTML report for all samples on the run
    • wf-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
    • <SAMPLE_NAME>.depth.txt: samtools depth, columns are contig, postion, and coverage

Tags

#workflows#nextflow
Matt Parker

Matt Parker

Clinical Bioinformatics Lead

Table Of Contents

1
How To Run
2
Data Analysis Details
3
Useful Links

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