Apr 07, 2020
Version 4
SARS-CoV-2 Enrichment Sequencing by Spiked Primer MSSPE method V.4
- Jessica E. Manning1,
- Jennifer Bohl2,
- Sreyngim Lay1,
- Sophana Chea1,
- Vida Ahyong3,
- Erik Karlsson4
- 1National Institute for Allergy and Infectious Diseases;
- 2National Institute of Allergy and Infectious Diseases;
- 3CZ Biohub;
- 4Institut Pasteur Cambodge
- Chan Zuckerberg Biohub
Protocol Citation: Jessica E. Manning, Jennifer Bohl, Sreyngim Lay, Sophana Chea, Vida Ahyong, Erik Karlsson 2020. SARS-CoV-2 Enrichment Sequencing by Spiked Primer MSSPE method. protocols.io https://dx.doi.org/10.17504/protocols.io.beshjeb6
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: April 07, 2020
Last Modified: April 07, 2020
Protocol Integer ID: 35369
Abstract
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.
This approach was developed based on the work from Deng et al, Nature Microbiology, January 13, 2020. https://www.nature.com/articles/s41564-019-0637-9
Materials
MATERIALS
NEBNext Ultra II RNA Library Prep Kit for Illumina - 96 rxnsNew England BiolabsCatalog #E7770L
NEBNext Adaptor for IlluminaNew England Biolabs
Qubit dsDNA HS Assay kit Thermo Fisher ScientificCatalog #Q32854
QIAamp Viral RNA Mini KitQiagenCatalog #52904
NEBNext USER EnzymeNew England BiolabsCatalog #E7458
Capillary electrophoresis instrument (e.g. Agilent Tapestation 4200)
High Sensitivity D5000 ScreenTapeAgilent TechnologiesCatalog #5067-5592
Qubit RNA HS Assay KitThermo Fisher ScientificCatalog #Q32852
TruSeq i7/i5 Indexing Primers - Custom (or NEBNext® Multiplex Oligos for Illumina)New England BiolabsCatalog #E7500L
ERCC RNA Spike-In MixThermo FisherCatalog #4456740
QIAseq FastSelect rRNA Removal KitQiagenCatalog #333180
DNase I SetZymo ResearchCatalog #E1010
STEP MATERIALS
QIAamp® Viral RNA Mini QiagenCatalog #52906
DNase I SetZymo ResearchCatalog #E1010
Qubit RNA HS Assay KitThermo Fisher ScientificCatalog #Q32852
High Sensitivity D5000 ScreenTapeAgilent TechnologiesCatalog #5067-5592
NEBNext Ultra II RNA Library Prep Kit for Illumina - 96 rxnsNew England BiolabsCatalog #E7770L
ERCC RNA Spike-In MixThermo FisherCatalog #4456740
QIAseq FastSelect rRNA Removal KitQiagenCatalog #333180
Protocol materials
NEBNext Adaptor for IlluminaNew England Biolabs
DNase I SetZymo ResearchCatalog #E1010
QIAseq FastSelect rRNA Removal KitQiagenCatalog #333180
DNase I SetZymo ResearchCatalog #E1010
High Sensitivity D5000 ScreenTapeAgilent TechnologiesCatalog #5067-5592
Capillary electrophoresis instrument (e.g. Agilent Tapestation 4200)
High Sensitivity D5000 ScreenTapeAgilent TechnologiesCatalog #5067-5592
Qubit RNA HS Assay KitThermo Fisher ScientificCatalog #Q32852
TruSeq i7/i5 Indexing Primers - Custom (or NEBNext® Multiplex Oligos for Illumina)New England BiolabsCatalog #E7500L
NEBNext Ultra II RNA Library Prep Kit for Illumina - 96 rxnsNew England BiolabsCatalog #E7770L
QIAamp® Viral RNA Mini QiagenCatalog #52906
NEBNext Ultra II RNA Library Prep Kit for Illumina - 96 rxnsNew England BiolabsCatalog #E7770L
ERCC RNA Spike-In MixThermo FisherCatalog #4456740
NEBNext USER EnzymeNew England BiolabsCatalog #E7458
QIAseq FastSelect rRNA Removal KitQiagenCatalog #333180
Qubit RNA HS Assay KitThermo Fisher ScientificCatalog #Q32852
Qubit dsDNA HS Assay kit Thermo Fisher ScientificCatalog #Q32854
QIAamp Viral RNA Mini KitQiagenCatalog #52904
ERCC RNA Spike-In MixThermo FisherCatalog #4456740
QIAamp® Viral RNA Mini QiagenCatalog #52906
DNase I SetZymo ResearchCatalog #E1010
Qubit RNA HS Assay KitThermo Fisher ScientificCatalog #Q32852
ERCC RNA Spike-In MixThermo FisherCatalog #4456740
QIAseq FastSelect rRNA Removal KitQiagenCatalog #333180
NEBNext Ultra II RNA Library Prep Kit for Illumina - 96 rxnsNew England BiolabsCatalog #E7770L
High Sensitivity D5000 ScreenTapeAgilent TechnologiesCatalog #5067-5592
Sample Collection
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
RNA extraction
Extraction of viral nucleic acids from clinical sample was performed with a QIAamp Viral RNA Mini Kit (Qiagen #52906) as described by manufacturer
QIAamp® Viral RNA Mini QiagenCatalog #52906
Extracted RNA samples were DNAse-treated using the Zymo DNAse I kit.
DNase I SetZymo ResearchCatalog #E1010
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 Quantification
RNA was quantified using the Qubit High Sensitivity RNA kit as described by the manufacturer.
Qubit RNA HS Assay KitThermo Fisher ScientificCatalog #Q32852
Qubit input RNA concentration for PUI = 6.9 ng/uL
Library Preparation
Library Preparation
Library Preparation was performed with the NEBNext Ultra II non-directional RNA kit.
NEBNext Ultra II RNA Library Prep Kit for Illumina - 96 rxnsNew England BiolabsCatalog #E7770L
Fragmentation & SARS-CoV-2 primer spike in
We 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.
| 1 reaction | ||
| Reagent | vol stock (uL) | |
| RNA (sample) (10ng – 100ng total) | 3.5 | |
| 25pg ERCC Spike-in (50pg/uL stock) * | 0.5 | |
| (pink) First SS Reaction Buffer 5x | 4 | |
| (pink) Random Primers/Spiked primer mix | 1 | |
| QIAseq FastSelect (1:100) rRNA** | 1 | |
| Total volume | 10 | |
| uL/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.
ERCC RNA Spike-In MixThermo FisherCatalog #4456740
** (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).
QIAseq FastSelect rRNA Removal KitQiagenCatalog #333180
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°C
- 2 min 70°C
- 2 min 65°C
- 2 min 60°C
- 2 min 55°C
- 5 min 37°C
- 5 min 25°C
The 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.
enrichmentprimers.pdf | Name | Sequence | |
| Primer_CoV1 | GTGACTTCCATGCCAATG | |
| Primer_CoV2 | CTGATTTTGGGGTCCATTATC | |
| Primer_CoV3 | GAAATGGTGAATTGCCCTC | |
| Primer_CoV4 | GATAGCAATTCCACCGGTG | |
| Primer_CoV5 | CAGTATAACCACCAATCTG | |
| Primer_CoV6 | CATTAATGCCAGAGATGTC | |
| Primer_CoV7 | GTATTTGTAATGCAGCAC | |
| Primer_CoV8 | CTTCTGTGCAGTTAACATC | |
| Primer_CoV9 | GATTCTGTTGGTTGGAC | |
| Primer_CoV10 | GAATGTAAAACTGAGGATCTG | |
| Primer_CoV11 | CAGCTGTACCTGGTGCAAC | |
| Primer_CoV12 | CACTACCTTCTGTAATAAG | |
| Primer_CoV13 | CATACAAACTGCCACCATC | |
| Primer_CoV14 | GTCCTTTGTACATAAGTG | |
| Primer_CoV15 | GAGCTGATTTGTCTTTATGTG | |
| Primer_CoV16 | CAGCATCACCATAGTCAC | |
| Primer_CoV17 | CGAACCGTTCAATCATAAG | |
| Primer_CoV18 | CACCATAGAATTTGCTTGTTC | |
| Primer_CoV19 | CTAGCTCTCTGAAGTGGTATC | |
| Primer_CoV20 | GTTTCTTCATGTTGGTAG | |
| Primer_CoV21 | CTAGCCCATTTCAAATCCTG | |
| Primer_CoV22 | GTTGTCCAGCATTTCTTCAC | |
| Primer_CoV23 | GACAAACTAGTATCAACCATATC | |
| Primer_CoV24 | CTGTCCTGGTTGAATGCGAAC | |
| Primer_CoV25 | CAGAGTACAGTGAATGAC | |
| Primer_CoV26 | GTAGATGCTATGTCACGAG | |
| Primer_CoV27 | GAACCTTTAGTGTTATTAG | |
| Primer_CoV28 | GTTCAAATAGCCTTCTCTG | |
| Primer_CoV29 | CTTAAAAGAGGGTGTGTAG | |
| Primer_CoV30 | CTCACCTACTGTCTTATTAC | |
| Primer_CoV31 | CATTTAGATCGTTAAGTGTG | |
| Primer_CoV32 | GTGCGAACAGTATCTACAC | |
| Primer_CoV33 | CACAACACAGGCGAACTC | |
| Primer_CoV34 | CACCTTCCTTAAACTTCTC | |
| Primer_CoV35 | CTTCTGAATTGTGACATGCTG | |
| Primer_CoV36 | GTCTCACCACTACGACCG | |
| Primer_CoV37 | GTTCACGGCAGCAGTATACACC | |
| Primer_CoV38 | TCCACAAAAGCACTTGTGGAAGC | |
| Primer_CoV39 | TGTGGGAAGTGTTTCTCCCTC | |
| Primer_CoV40 | GTCTGAACAACTGGTGTAAGTTCC | |
| Primer_CoV41 | ATTTCAGTAGTGCCACCAGCC | |
| Primer_CoV42 | CATGTCCACAACTTGCGTGTG | |
| Primer_CoV43 | AGCACCGTCTATGCAATACAAAG | |
| Primer_CoV44 | ACAGCAGCTAAACCATGAGTAGC | |
| Primer_CoV45 | ACAACCGTCTACAACATGCAC | |
| Primer_CoV46 | GTCACGGGGTGTCATGTTTTC | |
| Primer_CoV47 | CGTGTGTCAGGGCGTAAACTTTC | |
| Primer_CoV48 | GAGCCTTTGCGAGATGACAAC | |
| Primer_CoV49 | AACGGCAATTCCAGTTTGAGC | |
| Primer_CoV50 | GCGGTTGAGTAAACAAAAGAGGC | |
| Primer_CoV51 | GGGAACACAACCATCTCTTGC | |
| Primer_CoV52 | ACGATGCACCACCAAAGGATTC | |
| Primer_CoV53 | AATACCAGCATTTCGCATGGCA | |
| Primer_CoV54 | TAGCAGCATTACCATCCTGAGC | |
| Primer_CoV55 | TGCATTAACATTGGCCGTGAC | |
| Primer_CoV56 | ACAACCTGGAGCATTGCAAAC | |
| Primer_CoV57 | TCACATAGTGCATCAACAGCGG | |
| Primer_CoV58 | TAAAGTTGCCACATTCCTACGTGG | |
| Primer_CoV59 | TAACAAAGCACTCGTGGACAGC | |
| Primer_CoV60 | CCTGTTGTCCATCAAAGTGTCCC | |
| Primer_CoV61 | GATGAACCTGTTTGCGCATCTG | |
| Primer_CoV62 | CTATTTGTTCGCGTGGTTTGCC | |
| Primer_CoV63 | ACCCTGTTTTCCTTCAAGGTCC | |
| Primer_CoV64 | TGCTACCGGCCTGATAGATTTC | |
| Primer_CoV65 | TGCTGCATTCAGTTGAATCACC | |
| Primer_CoV66 | CAGAAGCTCTGATTTCTGCAGC | |
| Primer_CoV67 | TTGCAGTAGCGCGAACAAAATC | |
| Primer_CoV68 | ACGCACACAATCGAAGCGCAG | |
| Primer_CoV69 | TGCCAATCCTGTAGCGACTGTATGC | |
| Primer_CoV70 | AGGACACGGGTCATCAACTAC | |
| Primer_CoV71 | TGCCAGCCATTCTAGCAGGAG | |
| Primer_CoV72 | TGTGGTGGCTCTTTCAAGTCC | |
| Primer_CoV73 | TTTTGTCATTCTCCTAAGAAGC |
Enrichment primer sequences for SARS-CoV-2 genome
First Strand Synthesis
Mix the following by pipetting up and down.
| 1 rxn | ||
| Reagent | vol stock (uL) | |
| Fragmented & primed RNA | 10 | |
| Nuclease-free water | 8 | |
| (pink) NEBNext First Strand Synthesis Enzyme Mix | 2 | |
| Total volume | 20 uL/rxn | |
Thermocycler (heated lid set to 105°C):
- 10 mins at 25°C
- 15 mins at 42°C
- 15 mins at 70°C
- Hold at 4°C
Second Strand Synthesis
| 1 rxn | ||
| Reagent | vol stock (uL) | |
| First strand synthesized DNA | 20 | |
| (orange) 2nd SS Reaction buffer (10X) | 8 | |
| (orange) 2nd SS enzyme mix | 4 | |
| Nuclease-free water | 48 | |
| Total volume | 80 uL/rxn | |
Thermocycler (heated lid off):
- 1 hour at 16°C
- Hold at 4°C
SPRI cleanup
*allow beads to sit in RT for 30 mins prior
- Use 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 timess
- Air 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.
End Repair
Mix all the following by pipetting up and down
| 1 rxn | ||
| Reagent | vol stock (uL) | |
| Purified ds-cDNA | 50 | |
| (green) Ultra II End Prep reaction buffer (8.6x) | 7 | |
| (green) Ultra II End Prep enzyme mix | 3 | |
| Total volume | 60 uL/rxn | |
Thermocycler (heated lid set to 105°C):
- 30 mins at 20°C
- 30 mins at 65°C
- Hold at 4°C
Adapter Ligation
- dilute adaptor to dilution prior to making master mix. Adaptor concentration depends on the amount of input; 1:100 dilution for samples <5ng, 1:25 for input of >5ng
- Add adaptor separately after ligation master mix and ligation enhancer to avoid adaptor dimers.
| 1 rxn | ||
| Reagent | vol stock (uL) | |
| End Prep reaction mixture | 60 | |
| (red) NEBNext Ultra II ligation master mix | 30 | |
| (red) NEBNext ligation enhancer | 1 | |
| 1:100 Adaptor (Cat No. E7337AA) | 2.5 | |
| Total volume | 93.5 uL/rxn | |
Thermocycler (heated lid off):
- 15 mins at 20°C with heated lid off
- Proceed immediately to Bead Purification.
SPRI Cleanup
*allow beads to sit in RT for 30 mins prior
- Use 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.
USER/Q5 Indexing PCR
Mix 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.
| 1 rxn | ||
| Reagent | vol stock (ul) | |
| Purified, adaptor-ligated cDNA | 12** | |
| (white) USER Enzyme (Cat no. M5505L, 250uL) | 3 | |
| (blue) NEBNext Ultra II Q5 master mix | 25 | |
| 5uM i7 barcoded primer (NEB index primer/TruSeq/or similar)* | 5uL | |
| 5uM i5 barcoded primer (NEB Universal primer/TruSeq/or similar)* | 5uL | |
| Total volume | 50uL |
Cycling conditions:
| Thermocycler (heated lid at 105°C): | Cycles | |
| 37 °C for 15 mins | 1 | |
| 98 °C for 30s | 1 | |
| 98 °C for 10s | 6-15** | |
| 65 °C for 75s | ||
| 65 °C for 5 mins | 1 | |
| Hold at 4 °C |
** PCR cycles are dependent on the input RNA. For libraries with <5ng input, perform 15-19 cycles of PCR. For 5-20ng input, perform 10-14 cycles of PCR. For >20ng input, perform 6-8 cycles of PCR.
SPRI Cleanup
*allow beads to sit in RT for 30 mins prior
- Use 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
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.
High Sensitivity D5000 ScreenTapeAgilent TechnologiesCatalog #5067-5592
Expected result
Tapestation HS D5000 assay shows an average size library of 384bp at a concentration of 62.5 nM.
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
iSeq Loading
The Illumina iSeq was loaded with 20uL of a 100pM library with a 5% PhiX spike in.
Analyses
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.
Expected result
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.
Expected result