TITLE: Whole genome sequencing of H5N1 from dairy products with tiled 250bp amplicons 
AUTHORS: William C. Vuyk, Andrew Lail, Isla Emmen, Nura Hassan, Patrick Barros Tiburcio, Christina Newman, Nicholas R. Minor, Nancy Wilson, Thomas Friedrich, David O'Connor

[DESCRIPTION]
Here we present a new short tiled amplicon scheme for the whole-genome sequencing of H5N1 from dairy product samples, and the methods we have used to successfully obtain whole-genome H5N1 sequences from dairy product samples using these primers. The majority of these primers were created using primalscheme with recent H5N1 sequences provided by the Andersen Lab (https://github.com/andersen-lab/avian-influenza/tree/master/alignments) used as a reference.1-2 The primalscheme amplicons left coverage gaps at the very 5' and 3' ends of each gene segment, so we manually added additional primers to supplement these gaps informed by primers developed by the Moncla Lab (https://github.com/moncla-lab/h5-sequencing-protocol-dev/blob/main/RT_and_PCR_protocol.md).3 The attached BED file contains the primer sequences aligned to gene segment sequences from A/Bovine/texas/24-029328-01/2024(H5N1).4 We were inspired by the effectiveness of the QIAseq DIRECT SARS-CoV-2 250bp tiled amplicon scheme at amplifying difficult environmental samples for sequencing, and continue to use QIAseq Direct SARS-CoV-2 reagents and protocols in this workflow for that reason.5 We plan to add our qPCR and sequencing results from commercial dairy samples to our github at https://github.com/dholab/dairy-hpai-monitoring. 

[BEFORE_START]
The protocol below describes how we have generated whole-genome H5N1 sequences from commercial dairy products using a 250bp tiled amplicon scheme. The methods described are not the only way to sequence H5N1 with this primer set. We invite others to use these primers however they think will work best. Here we document what has worked best for us. 

[STEPS]
SECTION: Obtaining primer pools
1. Navigate to the oPools primer pools product page on IDT.
SECTION: Obtaining primer pools
2. To order, upload the   file attached to this protocol. There should be 178 valid sequences. Order at a concentration of 50pmol/oligo. 

SECTION: Obtaining primer pools
3. Purchase AVRL_H5N1_250bpAmpWGS_Pool1 and AVRL_H5N1_250bpAmpWGS_Pool2. Together, the two pools should cost about $240. At 50 pmol/oligo, this order should contain enough primers to amplify 96 samples in separate 25ul reactions for each pool. 

SECTION: Reverse transcription
6. Using reagents from the QIAseq DIRECT SARS-CoV-2 Kit A and QIAseq DIRECT SARS-CoV-2 Enhancer, follow the enhanced cDNA synthesis procedure starting on page 14 of the QIAseq DIRECT SARS-CoV-2 Handbook: .5

SECTION: Isolating, cleaning, and concentrating viral RNA from milk
4. See our other protocol RNA extraction from milk for HPAI surveillance. When preparing samples for this sequencing protocol, we recommend following the optional clean and concentration steps in the RNA extraction protocol. 
SECTION: Quantifying H5N1 RNA concentration
5. We recommend using qPCR or dPCR to determine if your isolated samples have H5N1 RNA, and if so, to quantify how much. We have used both an influenza A primer/probe set     and an H5 specific primer/probe set     to test dairy samples. See our dairy-hpai-monitoring Github repository for more PCR primers and probe sequences we have found to work. Our qPCR results can be seen in the DETECTION_RESULTS.tsv, and primer/probe fastas can be found in the repository's assets folder. We will also post sequencing results in the same Github repository, so look there as well for examples of what sample concentrations can be successfully sequenced with our method. 

SECTION: Sequencing Library Prep
10.  Below are instructions on how to sequence using either Illumina (Miseq V2 2x150) or Oxford Nanopore (Native Barcoding 96 V14) sequencing chemistries. 
SECTION: Reverse transcription
6.1. Following the QIAseq DIRECT SARS-CoV-2 Handbook, for each sample assemble a cDNA synthesis reaction in strip cap tubes or 96-well plate on ice as follows (or add RNA to an aliquoted master mix of reagents 2-6).  

 

SECTION: Reverse transcription
6.2. Following the QIAseq Direct SARS-CoV-2 Handbook cDNA synthesis incubation protocol (page 15), use a thermal cycler to perform the cDNA synthesis incubation with the following conditions:
 

SECTION: Primer pool preparation
7. Add nuclease-free water or 1xTE buffer to lyophilized AVRL_H5N1_250bpAmpWGS_Pool1 and AVRL_H5N1_250bpAmpWGS_Pool2 to create 100uM stocks.   
SECTION: Primer pool preparation
8. Dilute aliquots of 100uM stock 10x in nuclease free water to create 10uM working stock. You will need 4ul of each 10uM primer pool per sample, like in the nCoV-2019 sequencing protocol v3 (LoCost) V.3.7
SECTION: PCR amplification
9.2. Following the QIAseq Direct SARS-CoV-2 Handbook enrichment protocol 5a (page 17), use a thermal cycler to perform the PCR using the following conditions:

 

SECTION: PCR amplification
9.1. Following the QIAseq DIRECT SARS-CoV-2 Handbook (replacing SARS-CoV-2 primer pools with H5N1 primer pools), for each 10uM H5N1 primer pool (AVRL_H5N1_250bpAmpWGS_Pool1 and AVRL_H5N1_250bpAmpWGS_Pool2) set up the following reaction for each sample in strip caps or a 96-well plate on ice:
 

SECTION: PCR amplification
9. Using prepared 10uM H5N1 primer pools from above and reagents from the QIAseq DIRECT SARS-CoV-2 Kit A and QIAseq DIRECT SARS-CoV-2 Enhancer, follow the enhanced SARS-CoV-2 enrichment procedure starting on page 16 of the QIAseq DIRECT SARS-CoV-2 Handbook. Use enrichment cycling protocol 5a.  .5
SECTION: Sequencing Library Prep
11. Pool the AVRL_H5N1_250bpAmpWGS_Pool1 and AVRL_H5N1_250bpAmpWGS_Pool2 25ul PCR products for each sample (for 50ul total per sample), mix, and determine sample concentration using a Qubit Fluorometer. 
SECTION: Sequencing Library Prep
12. Prepare for Oxford Nanopore sequencing following the Native Barcoding 96 V14 protocol   .8 Add 200fmol or 11.5ul of sample into the End Prep step. We have not had problems loading more than 200fmol. 

SECTION: Sequencing Library Prep
13. Load end-prepped, barcoded, and adapted sample library onto an R.10.4.1 flow cell and sequence on an ONT MK1C/MK1B or Gridion instrument. Load 50fmol or 12ul of prepared library. We have not had any problems loading more than 50fmol.
SECTION: Sequencing Library Prep
11. Pool the AVRL_H5N1_250bpAmpWGS_Pool1 and AVRL_H5N1_250bpAmpWGS_Pool2 25ul PCR products for each sample (for 50ul total per sample), mix, and determine sample concentration using a Qubit Fluorometer. 
SECTION: Sequencing Library Prep
12. Take 45ul of each pooled sample forward into the QIAseq bead cleanup step described on page 18 of the QIAseq Direct SARS-CoV-2 Handbook   , and follow the procedure from there on as written.5

SECTION: Sequencing Library Prep
13. Load 8pmol normalized pooled library into Miseq V2 2x150 reagent cartridge and sequence on Miseq instrument.9