TITLE: SARS-CoV-2 Enrichment Sequencing by Spiked Primer MSSPE method
AUTHORS: Jessica E. Manning, Jennifer Bohl, Sreyngim Lay, Sophana Chea, Vida Ahyong, Erik Karlsson

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><div class = "justify" style = "text-align:left">This protocol was used to enrich for SARS-CoV2 sequencing reads from a confirmed COVID-19 swab sample and attain the full genome using an iSeq100. By using a spiked primer approach with 73 primers spanning the entire SARS-CoV2 genome, we were able to get an average of 15x genome coverage on an iSeq100 with 1.8 million paired end-reads. Here we overview all the steps, from sample extraction, library preparation with a spiked primer enrichment step, and sequencing on an iSeq100. The collaborative effort involved the Manning Lab, NIAID in Phnom Penh, Cambodia, Institut Pasteur Cambodge, Cambodia Ministry of Health, the Chan Zuckerberg Biohub, and the Chan Zuckerberg Initiative.</div></div><div class = "text-block"><div class = "justify" style = "text-align:left"></div></div><div class = "text-block"><div class = "justify" style = "text-align:left"><span>This approach was developed based on the work from Deng et al, Nature Microbiology, January 13, 2020. </span><a href="https://www.nature.com/articles/s41564-019-0637-9" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">https://www.nature.com/articles/s41564-019</span></a><a href="https://www.nature.com/articles/s41564-019-0637-9" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;:;:;">-</span></a><a href="https://www.nature.com/articles/s41564-019-0637-9" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">063</span></a><a href="https://www.nature.com/articles/s41564-019-0637-9" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">7-9</span></a></div></div></div>

[STEPS]
?. [Sample Collection]
Nasopharyngeal and oropharyngeal swabs (combined into one tube) were collected from a symptomatic patient meeting case definition for possible infection with SARS-CoV-2.
?. [RNA extraction]
Extraction of viral nucleic acids from clinical sample was performed with a QIAamp Viral RNA Mini Kit (Qiagen #52906) as described by manufacturerExtracted RNA samples were DNAse-treated using the Zymo DNAse I kit. The nucleic acid was tested via real-time polymerase chain reaction for COVID-19 using both the Drosden and HKU protocols published by the World Health Organization and confirmed positive for COVID-19 on January 27th, 2020. Ct values for PUI =  24.
?. [RNA Quantification]
RNA was quantified using the Qubit High Sensitivity RNA kit as described by the manufacturer. Qubit input RNA concentration for PUI = 6.9 ng/uL
?. [Library Preparation]
Library Preparation was performed with the NEBNext Ultra II non-directional RNA kit.Fragmentation & SARS-CoV-2 primer spike inWe incorrectly believed the random primers were at a concentration of 10 μM.According to NEB, random primers in the NEBNext Ultra II non-directional library preparation protocol are at a concentration of 1.9mM. We had a successful enrichment by adding a 1:1 volume of 100 μM SARS-CoV-2 primers, an approximate 20:1 ratio of random primers to CoV primers. We have not yet tried other ratios.  AB11 reaction2Reagentvol stock (uL)3RNA (sample) (10ng – 100ng total)3.5425pg ERCC Spike-in (50pg/uL stock) *0.55(pink) First SS Reaction Buffer 5x46(pink) Random Primers/Spiked primer mix17QIAseq FastSelect (1:100) rRNA**18Total volume109uL/rxn* (optional) ERCCs are internal synthetic RNA controls comprised of 92 synthetic RNAs that do not match to any known microbe in the NCBI NR/NT databases. **  (optional) FastSelect is designed for removing human rRNA, omit this reagent if not using human derived samples and only incubate at 94°C and then directly to 4°C (omit ramping down steps). Thermocycler (heated lid set to 105°C):8 min at 94°C (Adjust this fragmentation time depending on the quality of extracted RNA)2 min 75°C2 min 70°C2 min 65°C2 min 60°C2 min 55°C5 min 37°C5 min 25°CThe 73 primers tile across the entire SARS-CoV2 genome at a spacing of ~400bp.  The PDF below shows the approximate location of the primer binding sites.  AB1NameSequence2Primer_CoV1GTGACTTCCATGCCAATG3Primer_CoV2CTGATTTTGGGGTCCATTATC4Primer_CoV3GAAATGGTGAATTGCCCTC5Primer_CoV4GATAGCAATTCCACCGGTG6Primer_CoV5CAGTATAACCACCAATCTG7Primer_CoV6CATTAATGCCAGAGATGTC8Primer_CoV7GTATTTGTAATGCAGCAC9Primer_CoV8CTTCTGTGCAGTTAACATC10Primer_CoV9GATTCTGTTGGTTGGAC11Primer_CoV10GAATGTAAAACTGAGGATCTG12Primer_CoV11CAGCTGTACCTGGTGCAAC13Primer_CoV12CACTACCTTCTGTAATAAG14Primer_CoV13CATACAAACTGCCACCATC15Primer_CoV14GTCCTTTGTACATAAGTG16Primer_CoV15GAGCTGATTTGTCTTTATGTG17Primer_CoV16CAGCATCACCATAGTCAC18Primer_CoV17CGAACCGTTCAATCATAAG19Primer_CoV18CACCATAGAATTTGCTTGTTC20Primer_CoV19CTAGCTCTCTGAAGTGGTATC21Primer_CoV20GTTTCTTCATGTTGGTAG22Primer_CoV21CTAGCCCATTTCAAATCCTG23Primer_CoV22GTTGTCCAGCATTTCTTCAC24Primer_CoV23GACAAACTAGTATCAACCATATC25Primer_CoV24CTGTCCTGGTTGAATGCGAAC26Primer_CoV25CAGAGTACAGTGAATGAC27Primer_CoV26GTAGATGCTATGTCACGAG28Primer_CoV27GAACCTTTAGTGTTATTAG29Primer_CoV28GTTCAAATAGCCTTCTCTG30Primer_CoV29CTTAAAAGAGGGTGTGTAG31Primer_CoV30CTCACCTACTGTCTTATTAC32Primer_CoV31CATTTAGATCGTTAAGTGTG33Primer_CoV32GTGCGAACAGTATCTACAC34Primer_CoV33CACAACACAGGCGAACTC35Primer_CoV34CACCTTCCTTAAACTTCTC36Primer_CoV35CTTCTGAATTGTGACATGCTG37Primer_CoV36GTCTCACCACTACGACCG38Primer_CoV37GTTCACGGCAGCAGTATACACC39Primer_CoV38TCCACAAAAGCACTTGTGGAAGC40Primer_CoV39TGTGGGAAGTGTTTCTCCCTC41Primer_CoV40GTCTGAACAACTGGTGTAAGTTCC42Primer_CoV41ATTTCAGTAGTGCCACCAGCC43Primer_CoV42CATGTCCACAACTTGCGTGTG44Primer_CoV43AGCACCGTCTATGCAATACAAAG45Primer_CoV44ACAGCAGCTAAACCATGAGTAGC46Primer_CoV45ACAACCGTCTACAACATGCAC47Primer_CoV46GTCACGGGGTGTCATGTTTTC48Primer_CoV47CGTGTGTCAGGGCGTAAACTTTC49Primer_CoV48GAGCCTTTGCGAGATGACAAC50Primer_CoV49AACGGCAATTCCAGTTTGAGC51Primer_CoV50GCGGTTGAGTAAACAAAAGAGGC52Primer_CoV51GGGAACACAACCATCTCTTGC53Primer_CoV52ACGATGCACCACCAAAGGATTC54Primer_CoV53AATACCAGCATTTCGCATGGCA55Primer_CoV54TAGCAGCATTACCATCCTGAGC56Primer_CoV55TGCATTAACATTGGCCGTGAC57Primer_CoV56ACAACCTGGAGCATTGCAAAC58Primer_CoV57TCACATAGTGCATCAACAGCGG59Primer_CoV58TAAAGTTGCCACATTCCTACGTGG60Primer_CoV59TAACAAAGCACTCGTGGACAGC61Primer_CoV60CCTGTTGTCCATCAAAGTGTCCC62Primer_CoV61GATGAACCTGTTTGCGCATCTG63Primer_CoV62CTATTTGTTCGCGTGGTTTGCC64Primer_CoV63ACCCTGTTTTCCTTCAAGGTCC65Primer_CoV64TGCTACCGGCCTGATAGATTTC66Primer_CoV65TGCTGCATTCAGTTGAATCACC67Primer_CoV66CAGAAGCTCTGATTTCTGCAGC68Primer_CoV67TTGCAGTAGCGCGAACAAAATC69Primer_CoV68ACGCACACAATCGAAGCGCAG70Primer_CoV69TGCCAATCCTGTAGCGACTGTATGC71Primer_CoV70AGGACACGGGTCATCAACTAC72Primer_CoV71TGCCAGCCATTCTAGCAGGAG73Primer_CoV72TGTGGTGGCTCTTTCAAGTCC74Primer_CoV73TTTTGTCATTCTCCTAAGAAGC
AB11 reaction2Reagentvol stock (uL)3RNA (sample) (10ng – 100ng total)3.5425pg ERCC Spike-in (50pg/uL stock) *0.55(pink) First SS Reaction Buffer 5x46(pink) Random Primers/Spiked primer mix17QIAseq FastSelect (1:100) rRNA**18Total volume109uL/rxn
AB1NameSequence2Primer_CoV1GTGACTTCCATGCCAATG3Primer_CoV2CTGATTTTGGGGTCCATTATC4Primer_CoV3GAAATGGTGAATTGCCCTC5Primer_CoV4GATAGCAATTCCACCGGTG6Primer_CoV5CAGTATAACCACCAATCTG7Primer_CoV6CATTAATGCCAGAGATGTC8Primer_CoV7GTATTTGTAATGCAGCAC9Primer_CoV8CTTCTGTGCAGTTAACATC10Primer_CoV9GATTCTGTTGGTTGGAC11Primer_CoV10GAATGTAAAACTGAGGATCTG12Primer_CoV11CAGCTGTACCTGGTGCAAC13Primer_CoV12CACTACCTTCTGTAATAAG14Primer_CoV13CATACAAACTGCCACCATC15Primer_CoV14GTCCTTTGTACATAAGTG16Primer_CoV15GAGCTGATTTGTCTTTATGTG17Primer_CoV16CAGCATCACCATAGTCAC18Primer_CoV17CGAACCGTTCAATCATAAG19Primer_CoV18CACCATAGAATTTGCTTGTTC20Primer_CoV19CTAGCTCTCTGAAGTGGTATC21Primer_CoV20GTTTCTTCATGTTGGTAG22Primer_CoV21CTAGCCCATTTCAAATCCTG23Primer_CoV22GTTGTCCAGCATTTCTTCAC24Primer_CoV23GACAAACTAGTATCAACCATATC25Primer_CoV24CTGTCCTGGTTGAATGCGAAC26Primer_CoV25CAGAGTACAGTGAATGAC27Primer_CoV26GTAGATGCTATGTCACGAG28Primer_CoV27GAACCTTTAGTGTTATTAG29Primer_CoV28GTTCAAATAGCCTTCTCTG30Primer_CoV29CTTAAAAGAGGGTGTGTAG31Primer_CoV30CTCACCTACTGTCTTATTAC32Primer_CoV31CATTTAGATCGTTAAGTGTG33Primer_CoV32GTGCGAACAGTATCTACAC34Primer_CoV33CACAACACAGGCGAACTC35Primer_CoV34CACCTTCCTTAAACTTCTC36Primer_CoV35CTTCTGAATTGTGACATGCTG37Primer_CoV36GTCTCACCACTACGACCG38Primer_CoV37GTTCACGGCAGCAGTATACACC39Primer_CoV38TCCACAAAAGCACTTGTGGAAGC40Primer_CoV39TGTGGGAAGTGTTTCTCCCTC41Primer_CoV40GTCTGAACAACTGGTGTAAGTTCC42Primer_CoV41ATTTCAGTAGTGCCACCAGCC43Primer_CoV42CATGTCCACAACTTGCGTGTG44Primer_CoV43AGCACCGTCTATGCAATACAAAG45Primer_CoV44ACAGCAGCTAAACCATGAGTAGC46Primer_CoV45ACAACCGTCTACAACATGCAC47Primer_CoV46GTCACGGGGTGTCATGTTTTC48Primer_CoV47CGTGTGTCAGGGCGTAAACTTTC49Primer_CoV48GAGCCTTTGCGAGATGACAAC50Primer_CoV49AACGGCAATTCCAGTTTGAGC51Primer_CoV50GCGGTTGAGTAAACAAAAGAGGC52Primer_CoV51GGGAACACAACCATCTCTTGC53Primer_CoV52ACGATGCACCACCAAAGGATTC54Primer_CoV53AATACCAGCATTTCGCATGGCA55Primer_CoV54TAGCAGCATTACCATCCTGAGC56Primer_CoV55TGCATTAACATTGGCCGTGAC57Primer_CoV56ACAACCTGGAGCATTGCAAAC58Primer_CoV57TCACATAGTGCATCAACAGCGG59Primer_CoV58TAAAGTTGCCACATTCCTACGTGG60Primer_CoV59TAACAAAGCACTCGTGGACAGC61Primer_CoV60CCTGTTGTCCATCAAAGTGTCCC62Primer_CoV61GATGAACCTGTTTGCGCATCTG63Primer_CoV62CTATTTGTTCGCGTGGTTTGCC64Primer_CoV63ACCCTGTTTTCCTTCAAGGTCC65Primer_CoV64TGCTACCGGCCTGATAGATTTC66Primer_CoV65TGCTGCATTCAGTTGAATCACC67Primer_CoV66CAGAAGCTCTGATTTCTGCAGC68Primer_CoV67TTGCAGTAGCGCGAACAAAATC69Primer_CoV68ACGCACACAATCGAAGCGCAG70Primer_CoV69TGCCAATCCTGTAGCGACTGTATGC71Primer_CoV70AGGACACGGGTCATCAACTAC72Primer_CoV71TGCCAGCCATTCTAGCAGGAG73Primer_CoV72TGTGGTGGCTCTTTCAAGTCC74Primer_CoV73TTTTGTCATTCTCCTAAGAAGC
?. [Library Preparation]
First Strand SynthesisMix the following by pipetting up and down.   AB11 rxn2Reagentvol stock (uL)3Fragmented &amp; primed RNA104Nuclease-free water85(pink) NEBNext First Strand Synthesis Enzyme Mix26Total volume20 uL/rxn7 Thermocycler (heated lid set to 105°C):10 mins at 25°C15 mins at 42°C15 mins at 70°CHold at 4°C
AB11 rxn2Reagentvol stock (uL)3Fragmented &amp; primed RNA104Nuclease-free water85(pink) NEBNext First Strand Synthesis Enzyme Mix26Total volume20 uL/rxn7
?. [Library Preparation]
Second Strand Synthesis  AB11 rxn2Reagentvol stock (uL)3First strand synthesized DNA204(orange) 2nd SS Reaction buffer (10X)85(orange) 2nd SS enzyme mix46Nuclease-free water487Total volume80 uL/rxn8Thermocycler (heated lid off):1 hour at 16°C Hold at 4°C
AB11 rxn2Reagentvol stock (uL)3First strand synthesized DNA204(orange) 2nd SS Reaction buffer (10X)85(orange) 2nd SS enzyme mix46Nuclease-free water487Total volume80 uL/rxn8
?. [Library Preparation]
SPRI cleanup*allow beads to sit in RT for 30 mins priorUse SPRI Beads1.8x ratio of beads-to-total volume of sample. Prep 80% EtOH.Add 144uL of room temperature beads to 2nd Strand Synthesis Rxn. Mix well by pipetting gently.Pulse spin the tubes, but be sure not to spin down beads. Incubate for 5 mins at room temperature.Place samples on magnetic rack, and incubate for 5 mins on the rack.Remove supernatant.Add 200uL of 80% EtOH to samples while on the magnetic rack. Incubate at room temperature for 30s then remove the supernatant.Repeat EtOH wash step for a total of 2 timessAir dry the beads for 5 mins while on the magnetic rack.Remove tube from magnetic rack. Elute DNA from beads into 53uL of 0.1x TE Buffer, 10mM Tris-HCl, or Nuclease free water.Vortex to mix. Spin tubes and incubate for 2 mins at room temperature off the magnetic rack.Place on magnetic rack until solution is clear ~ 5 mins.Remove 50uL of the supernatant and transfer to a clean nuclease free PCR tube.**Checkpoint: Samples can be stored frozen at -20 °C and library prep resumed the next day.
?. [Library Preparation]
End RepairMix all the following by pipetting up and down  AB11 rxn2Reagentvol stock (uL)3Purified ds-cDNA504(green) Ultra II End Prep reaction buffer (8.6x)75(green) Ultra II End Prep enzyme mix36Total volume60 uL/rxn7 Thermocycler (heated lid set to 105°C):30 mins at 20°C30 mins at 65°CHold at 4°C
AB11 rxn2Reagentvol stock (uL)3Purified ds-cDNA504(green) Ultra II End Prep reaction buffer (8.6x)75(green) Ultra II End Prep enzyme mix36Total volume60 uL/rxn7
?. [Library Preparation]
Adapter Ligationdilute adaptor to dilution prior to making master mix. Adaptor concentration depends on the amount of input; 1:100 dilution for samples 5ngAdd adaptor separately after ligation master mix and ligation enhancer to avoid adaptor dimers.uL/rxn Thermocycler (heated lid off):15 mins at 20°C with heated lid offProceed immediately to Bead Purification.   AB11 rxn2Reagentvol stock (uL)3End Prep reaction mixture604(red) NEBNext Ultra II ligation master mix305(red) NEBNext ligation enhancer161:100 Adaptor (Cat No. E7337AA)2.57Total volume93.5 uL/rxn8 Thermocycler (heated lid off):15 mins at 20°C with heated lid offProceed immediately to Bead Purification.
AB11 rxn2Reagentvol stock (uL)3End Prep reaction mixture604(red) NEBNext Ultra II ligation master mix305(red) NEBNext ligation enhancer161:100 Adaptor (Cat No. E7337AA)2.57Total volume93.5 uL/rxn8
?. [Library Preparation]
SPRI Cleanup*allow beads to sit in RT for 30 mins priorUse SPRI bead 0.9x ratio of beads-to-total volume of sample. Prep 80% EtOH.Add 87uL of room temperature beads (0.9x) to Adaptor Ligation reaction. Mix well.Pulse spin the tubes, but be sure not to spin down beads. Incubate for 5 mins at room temperature.Place samples on magnetic rack, and incubate for 5 mins on the rack.Remove supernatant.Add 200uL of 80% EtOH to samples while on the magnetic rack. Incubate at room temperature for 30s then remove the supernatant.Repeat EtOH wash step for a total of 2 times.Air dry the beads for 5 mins while on the magnetic rack.Remove tube from magnetic rack. Elute DNA from beads into 17uL of 0.1x TE Buffer, 10mM Tris-HCl, or Nuclease free water.Vortex to mix. Spin tubes and incubate for 2 mins at room temperature off the magnetic rack.Place on magnetic rack until solution is clear ~ 5 mins.Remove 15uL of the supernatant and transfer to a clean nuclease free PCR tube.**Checkpoint: Samples can be stored frozen at -20 °C and library prep resumed the next day.
?. [Library Preparation]
USER/Q5 Indexing PCRMix the following components by pipetting up and down. *Technical Note from NEB: The TruSeq adaptor and primer strategy from Illumina uses a barcoded adaptor and universal primer. If someone used the universal NEBNext adaptor and Illumina TruSeq universal primer, the result would be a lack of amplification. It is of course possible to use non- NEB adaptors and primers. We support it and we do have an FAQ about this: https://www.neb.com/faqs/2019/03/08/can-i-use-this-nebnext-kit-with-adaptors-and-primers-from-other-vendors-than-neb ** In this new version, we have reduced the purified, adaptor-ligated cDNA to 12uL rather than 15uL to assure that the reaction is not underbuffered. Alternatively, the previous SPRI reaction can concentrate the cDNA further prior to setting up the PCR reaction.  AB1    1 rxn2    Reagentvol stock (ul)3Purified, adaptor-ligated cDNA12**4(white) USER Enzyme (Cat no. M5505L,
  250uL)35(blue) NEBNext Ultra II Q5 master mix2565uM i7 barcoded primer (NEB index
  primer/TruSeq/or similar)*5uL75uM i5 barcoded primer (NEB Universal
  primer/TruSeq/or similar)*5uL8Total volume50uLCycling conditions:  AB1Thermocycler (heated lid at 105°C):Cycles237 °C for 15 mins1398 °C for 30s1498 °C for 10s6-15**565 °C for 75s665 °C for 5 mins17Hold at 4 °C ** PCR cycles are dependent on the input RNA. For libraries with 20ng input, perform 6-8 cycles of PCR.
AB1    1 rxn2    Reagentvol stock (ul)3Purified, adaptor-ligated cDNA12**4(white) USER Enzyme (Cat no. M5505L,
  250uL)35(blue) NEBNext Ultra II Q5 master mix2565uM i7 barcoded primer (NEB index
  primer/TruSeq/or similar)*5uL75uM i5 barcoded primer (NEB Universal
  primer/TruSeq/or similar)*5uL8Total volume50uL
AB1Thermocycler (heated lid at 105°C):Cycles237 °C for 15 mins1398 °C for 30s1498 °C for 10s6-15**565 °C for 75s665 °C for 5 mins17Hold at 4 °C
?. [Library Preparation]
SPRI Cleanup*allow beads to sit in RT for 30 mins priorUse SPRI Beads at 0.8x ratio of beads-to-total volume of sample. Prep 80% EtOH.Add 43uL of room temperature Ampure Beads (0.8x) to barcoded DNA. Mix well.Pulse spin the tubes, but be sure not to spin down beads. Incubate for 5 mins at room temperature.Place samples on magnetic rack, and incubate for 5 mins on the rack.Remove supernatant.Add 200uL of 80% EtOH to samples while on the magnetic rack. Incubate at room temperature for 30s then remove the supernatant.Repeat EtOH wash step for a total of 2 times.Air dry the beads for 5 mins while on the magnetic rack.Remove tube from magnetic rack. Elute DNA from beads into 23uLof 0.1x TE Buffer, 10mMTris-HCL, or Nuclease free water.Vortex to mix. Spin tubes and incubate for 2 mins at room temperature off the magnetic rack.Place on magnetic rack until solution is clear ~ 5 mins.Remove 20uL of the supernatant and transfer to a clean nuclease free PCR tube.** Checkpoint: Libraries are complete, samples can be stored frozen at -20 °C until ready for quantification and pooling.
?. [Library Quality Control]
Libraries were quantified by the Qubit High Sensitivity DNA kit and the Agilent High Sensitivity D500 DNA Tapestation assay. If adapter dimers are found, library should be size selected using a SPRI ratio of 0.8x. Quantified library is then diluted down to the loading concentration for the iSeq, 100pM. This value will vary depending on the type of sequencer.
?. [iSeq Loading]
The Illumina iSeq was loaded with 20uL of a 100pM library with a 5% PhiX spike in.
?. [Analyses]
Metagenomic sequencing results were uploaded onto IDseq.net directly from the Illumina Basespace Sequence Hub. The open-source cloud-based pipeline analyzed 1.8 million reads. The majority of reads mapped to the nasal and oral microbiome. Analyses of the viral components resulted in 3,077 single end reads aligning to the SARS-CoV2 taxon. Resulting reads from the Betacoronovirus genus were downloaded from IDseq and mapped to the NCBI genome accession number: MN908947.1. The geneious alignment displayed even coverage across the genome with an average coverage of 14.9x. One SNP was noted at position 25,654 in ORF3a resulting in a valine to leucine substitution when compared to NCBI accession MN908947.1.