Apr 11, 2020
A protocol for massively parallel diagnosis and genome sequencing of SARS-CoV-2
- Leigh G Monahan1,
- Kay Anantanawat1,
- Joyce To1,
- Aaron Darling1
- 1University of Technology Sydney
- Coronavirus Method Development Community
Protocol Citation: Leigh G Monahan, Kay Anantanawat, Joyce To, Aaron Darling 2020. A protocol for massively parallel diagnosis and genome sequencing of SARS-CoV-2. protocols.io https://dx.doi.org/10.17504/protocols.io.betrjem6
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: In development
This protocol is currently in the early stages of testing. Feedback and suggestions are welcomed.
Created: April 08, 2020
Last Modified: April 11, 2020
Protocol Integer ID: 35409
Abstract
Managing the current COVID-19 pandemic requires diagnostic testing at an unprecedented scale. However, the crisis has revealed severe deficiencies in our capacity to perform such testing. Here we build on the work of others (1-3) to develop a protocol that not only enables many thousand diagnostic tests to be run in parallel, but also provides near-whole genome sequencing data to facilitate phylogenetic analysis and contact tracing. One of the key features of our protocol is a magnetic bead-based strategy for RNA capture that may circumvent the need for SARS-CoV-2 RNA extraction, currently one of the major bottlenecks in both reagent supply and hands-on sample processing time. This approach also eliminates the requirement for per-sample reverse transcription, significantly reducing per-sample costs.
The major steps in our wet lab workflow can be summarised as follows. First, we generate a collection of bead-bound, single-stranded DNA probes tiling the entire SARS-CoV-2 genome. Multiple uniquely barcoded probe sets are prepared, each of which are used to capture viral RNA directly from patient swab samples via DNA/RNA hybridisation. Samples are combined together at this stage, enabling cDNA synthesis to be performed in a single pooled reaction. Finally, multiplex PCR is used to generate a library of overlapping amplicons ready for Illumina sequencing.
A schematic overview of the workflow is attached below, along with a more detailed figure depicting the various stages of library preparation.
References
Materials
Reagents
| ITEM | SUPPLIER | CATALOGUE NUMBER | |
| General consumables, chemicals and equipment | |||
| UltraPure 1M Tris-HCl pH 7.5 | Invitrogen | 15567027 | |
| EDTA | Sigma-Aldrich | EDS-100G | |
| NaCl | Sigma-Aldrich | S3014-500G | |
| UltraPure DNase/RNase-free distilled water (500 mL) | Invitrogen | 10977015 | |
| Ethanol, 200 proof for molecular biology (500 mL) | Sigma-Aldrich | E7023-500mL | |
| DNA LoBind tubes, 1.5 mL (250 tubes) | Eppendorf | 30108051 | |
| 0.2 mL MAXYMum Recovery Thin Wall PCR Tubes | Fisher Biotec | PCR-02-L-C | |
| 20X TE buffer - RNase-free | Invitrogen | T11493 | |
| Sodium citrate | Sigma-Aldrich | C8532-100G | |
| NaOH | Sigma-Aldrich | S8045-500G | |
| PCR plate - Twintec LoBind DNA (Eppendorf) | Sigma-Aldrich | EP0030129512-25EA | |
| LiCl | Sigma-Aldrich | L9650-100G | |
| LiDS | Sigma-Aldrich | L9781-5G | |
| DTT | Sigma-Aldrich | D9779-250MG | |
| PCR foil | Eppendorf | 30127790 | |
| Step 1: Preparation of barcoded beads for RNA capture | |||
| Dynabeads MyOne Streptavidin C1 | Invitrogen | 65001 | |
| Custom DNA oligos | IDT | - | |
| 2X B&W buffer | See Buffer list | - | |
| 1X SSC buffer | See Buffer list | - | |
| PrimeSTAR GXL Polymerase | TaKaRa | R050A | |
| Step 2: Sample collection and preparation | |||
| Universal Transport Medium (UTM) | COPAN | 306C | |
| Lysis/binding buffer | See Buffer list | - | |
| Qubit HS RNA Assay Kit | Invitrogen | Q32855 | |
| Qubit tubes | Invitrogen | Q32856 | |
| Step 3: RNA capture | |||
| Washing buffer A | See Buffer list | - | |
| Step 4: cDNA synthesis | |||
| SuperScript™ IV First-Strand Synthesis System | Invitrogen | 18091200 | |
| Washing buffer B | See Buffer List | - | |
| Step 5: Library amplification | |||
| Q5 Hot Start HiFi DNA Polymerase | NEB | M0493S | |
| Custom DNA oligos | IDT | - | |
| Qubit dsDNA HS Assay Kit | Invitrogen | Q33230 | |
| PrimeSTAR GXL Polymerase | TaKaRa | R050A | |
| SPRIselect beads | Beckman Coulter | B23318 | |
| Step 6: Library quantitation and sequencing | |||
| Bioanalyzer HS dsDNA | Agilent | 5067-4626 | |
| Qubit dsDNA HS | Invitrogen | Q33230 |
Buffer list
| BUFFER NAME | COMPONENTS | |
| 2X B&W buffer | 10 mM Tris-HCl (pH 7.5) | |
| 1 mM EDTA | ||
| 2 M NaCl | ||
| 1X SSC buffer | 0.15 M NaCl | |
| 0.015 M sodium citrate | ||
| Adjust pH to 7.0 with NaOH | ||
| TE buffer | 10 mM Tris-HCl (pH 7.5) | |
| 1 mM EDTA | ||
| Lysis/binding buffer | 100 mM Tris-HCl (pH 7.5) | |
| 500 mM LiCl | ||
| 10 mM EDTA, pH 8 | ||
| 1% LiDS | ||
| 5 mM dithiothreitol (DTT) | ||
| Washing buffer A | 10 mM Tris-HCl (pH 7.5) | |
| 150 mM LiCl | ||
| 1 mM EDTA | ||
| 0.1% LiDS | ||
| Washing buffer B | 10 mM Tris-HCl (pH 7.5) | |
| 150mM LiCl |
Individual primers [version 1]
| NAME | SEQUENCE | |
| [Biotin]-barcoded bead tether_1 | /5biosg/TCTAGAGCCACCAGCGGCATAGTAATCGTCGGCAGCGTCAGATGTGTATAAGAGACAGGAACGTAATCGGCTCGGGTCGTCTG | |
| [Biotin]-barcoded bead tether_2 | /5biosg/TCTAGAGCCACCAGCGGCATAGTAATCGTCGGCAGCGTCAGATGTGTATAAGAGACAGATTGATTACGGGCTCGGGTCGTCTG | |
| [Biotin]-barcoded bead tether_3 | /5biosg/TCTAGAGCCACCAGCGGCATAGTAATCGTCGGCAGCGTCAGATGTGTATAAGAGACAGTCCGTCATCTGGCTCGGGTCGTCTG | |
| [Biotin]-barcoded bead tether_4 | /5biosg/TCTAGAGCCACCAGCGGCATAGTAATCGTCGGCAGCGTCAGATGTGTATAAGAGACAGACTAGTTGCGGGCTCGGGTCGTCTG | |
| [Biotin]-barcoded bead tether_5 | /5biosg/TCTAGAGCCACCAGCGGCATAGTAATCGTCGGCAGCGTCAGATGTGTATAAGAGACAGACCATATAAGGGCTCGGGTCGTCTG | |
| [Biotin]-barcoded bead tether_6 | /5biosg/TCTAGAGCCACCAGCGGCATAGTAATCGTCGGCAGCGTCAGATGTGTATAAGAGACAGATCTCGATGAGGCTCGGGTCGTCTG | |
| [Biotin]-barcoded bead tether_7 | /5biosg/TCTAGAGCCACCAGCGGCATAGTAATCGTCGGCAGCGTCAGATGTGTATAAGAGACAGACTTATAGTTGGCTCGGGTCGTCTG | |
| [Biotin]-bead tether | /5biosg/TCTAGAGCCACCAGCGGCATAGTAATCGTCGGCAGCGTC | |
| Barcoded linker_1 | CAGACGACCCGAGCCTACCATGCTTCTGTCTCTTATACACATCTGACGCTGCCGACGA | |
| Barcoded linker_2 | CAGACGACCCGAGCCAATGGAGCCGCTGTCTCTTATACACATCTGACGCTGCCGACGA | |
| Barcoded linker_3 | CAGACGACCCGAGCCCTAATCCGGTCTGTCTCTTATACACATCTGACGCTGCCGACGA | |
| Barcoded linker_4 | CAGACGACCCGAGCCTGCCTAACTCCTGTCTCTTATACACATCTGACGCTGCCGACGA | |
| Barcoded linker_5 | CAGACGACCCGAGCCAATAGTAGGCCTGTCTCTTATACACATCTGACGCTGCCGACGA | |
| Barcoded linker_6 | CAGACGACCCGAGCCGCAACTTGGACTGTCTCTTATACACATCTGACGCTGCCGACGA | |
| Barcoded linker_7 | CAGACGACCCGAGCCGAGATCGTCACTGTCTCTTATACACATCTGACGCTGCCGACGA | |
| Illumina P5 index primer_1 | AATGATACGGCGACCACCGAGATCTACACTCGCTGAATCGTCGGCAGCGTC | |
| Illumina P5 index primer_2 | AATGATACGGCGACCACCGAGATCTACACCTCCAGGATCGTCGGCAGCGTC | |
| Illumina P7 index primer_1 | CAAGCAGAAGACGGCATACGAGATAACCGCGGGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT | |
| Illumina P7 index primer_2 | CAAGCAGAAGACGGCATACGAGATGGTTATAAGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT | |
| Illumina P5 flow cell primer | AATGATACGGCGACCACCGAGATCTACAC |
SARS-CoV-2 probe pool 1 [version 1]
| NAME | SEQUENCE | |
| ncov_cap_1_RIGHT | CAGCACATCTAGGTTTCGTCCGCAGACGACCCGAGCC | |
| ncov_cap_3_RIGHT | CCTCATGGTCATGTTATGGTTGAGCCAGACGACCCGAGCC | |
| ncov_cap_5_RIGHT | GTTACCCGTGAACTCATGCGTGCAGACGACCCGAGCC | |
| ncov_cap_7_RIGHT | GCAGACACCTTTTGAAATTAAATTGGCCAGACGACCCGAGCC | |
| ncov_cap_9_RIGHT | GGTGAAACTTCATGGCAGACGGCAGACGACCCGAGCC | |
| ncov_cap_11_RIGHT | GTAAGGGTGGTCGCACTATTGCCAGACGACCCGAGCC | |
| ncov_cap_13_RIGHT | CTGCTTCCACAAGTGCTTTTGTCAGACGACCCGAGCC | |
| ncov_cap_15_RIGHT | CGTGTTTTACAGAAGGCCGCTACAGACGACCCGAGCC | |
| ncov_cap_17_RIGHT | AGAGACGGTTGGGAAATTGTTAAATTTCAGACGACCCGAGCC | |
| ncov_cap_19_RIGHT | AGAAGAAACTGGCCTACTCATGCCAGACGACCCGAGCC | |
| ncov_cap_21_RIGHT | TGGTAACAAACAATACCTTCACACTCACAGACGACCCGAGCC | |
| ncov_cap_23_RIGHT | ACCACTGGGCATTGATTTAGATGACAGACGACCCGAGCC | |
| ncov_cap_25_RIGHT | AGAGCAAGAAGAAGATTGGTTAGATGACAGACGACCCGAGCC | |
| ncov_cap_27_RIGHT | GTGGTTGTTAATGCAGCCAATGTCAGACGACCCGAGCC | |
| ncov_cap_29_RIGHT | CAGCACGAAGTTCTACTTGCACCAGACGACCCGAGCC | |
| ncov_cap_31_RIGHT | ACCTTCAGTTGAACAGAGAAAACAAGCAGACGACCCGAGCC | |
| ncov_cap_33_RIGHT | AAAGGCTGGTGGCACTACTGAACAGACGACCCGAGCC | |
| ncov_cap_35_RIGHT | CAGAAGAAACACGCAAATTAATGCCCAGACGACCCGAGCC | |
| ncov_cap_37_RIGHT | AAGTGCCAGCTACAGTTTCTGTCAGACGACCCGAGCC | |
| ncov_cap_39_RIGHT | GTTATCACCTTTGACAATCTTAAGACACTCAGACGACCCGAGCC | |
| ncov_cap_41_RIGHT | TGATCCTAGTTTTCTGGGTAGGTACACAGACGACCCGAGCC | |
| ncov_cap_43_RIGHT | GAAGCTGCTAACTTTTGTGCACTCAGACGACCCGAGCC | |
| ncov_cap_45_RIGHT | CGTGTGGTAAACAAGCTACAAAATATCTCAGACGACCCGAGCC | |
| ncov_cap_47_RIGHT | CAAAGGTCCTATTACGGATGTTTTCTACCAGACGACCCGAGCC | |
| ncov_cap_49_RIGHT | ACATTTTTCCCTGACTTAAATGGTGATGCAGACGACCCGAGCC | |
| ncov_cap_51_RIGHT | TGAAGAAGTAGTGGAAAATCCTACCATCAGACGACCCGAGCC | |
| ncov_cap_53_RIGHT | TGCTGTTAATAGTGTCCCTTGGGCAGACGACCCGAGCC | |
| ncov_cap_55_RIGHT | CTCAACCGCTGCTTTAGGTGTTCAGACGACCCGAGCC | |
| ncov_cap_57_RIGHT | TGGTTTTTGGCATATATTCTTTTCACTAGGCAGACGACCCGAGCC | |
| ncov_cap_59_RIGHT | TCAACTTGTATGATGTGTTACAAACGTAACAGACGACCCGAGCC | |
| ncov_cap_61_RIGHT | ACAGTGAAGAATGGTTCCATCCATCAGACGACCCGAGCC | |
| ncov_cap_63_RIGHT | TGTTACTAGATCAGGCATTAGTGTCTGCAGACGACCCGAGCC | |
| ncov_cap_65_RIGHT | AGGGTTTGTTGATTCAGATGTAGAAACCAGACGACCCGAGCC | |
| ncov_cap_67_RIGHT | ACGAAAACAAATACGTAGTGCTGCCAGACGACCCGAGCC | |
| ncov_cap_69_RIGHT | TGTCATGTCTAAACATACTGACTTTTCAAGCAGACGACCCGAGCC | |
| ncov_cap_71_RIGHT | ATTACGCACAACTAATGGTGACTTTTCAGACGACCCGAGCC | |
| ncov_cap_73_RIGHT | ACGTTATGTGCTCATGGATGGCCAGACGACCCGAGCC | |
| ncov_cap_75_RIGHT | GGTGCTTTGGACATATCAGCATCTCAGACGACCCGAGCC | |
| ncov_cap_77_RIGHT | AGCACATATTCAGTGGATGGTTATGTTCAGACGACCCGAGCC | |
| ncov_cap_79_RIGHT | TCTAAAGTTGCGTAGTGATGTGCTACAGACGACCCGAGCC | |
| ncov_cap_81_RIGHT | CCCATCTGGTAAAGTTGAGGGTTGCAGACGACCCGAGCC | |
| ncov_cap_83_RIGHT | GCTTAAGGTTGATACAGCCAATCCTCAGACGACCCGAGCC | |
| ncov_cap_85_RIGHT | TCTTTTTGTTACATGCACCATATGGAATTCAGACGACCCGAGCC | |
| ncov_cap_87_RIGHT | CCTCTAACACAAGACCATGTTGACACAGACGACCCGAGCC | |
| ncov_cap_89_RIGHT | CACACCACTGGTTGTTACTCACACAGACGACCCGAGCC | |
| ncov_cap_91_RIGHT | TGCTAGTTGGGTGATGCGTATTCAGACGACCCGAGCC | |
| ncov_cap_93_RIGHT | TCTCTGTTACTTCTAACTACTCAGGTGTACAGACGACCCGAGCC | |
| ncov_cap_95_RIGHT | ACTGACTCTTGGTGTTTATGATTACTTAGTCAGACGACCCGAGCC | |
| ncov_cap_97_RIGHT | GGGCTCAATGTGTCCAGTTACACAGACGACCCGAGCC | |
| ncov_cap_99_RIGHT | TGCTAATGGTGATTCTGAAGTTGTTCTCAGACGACCCGAGCC | |
| ncov_cap_101_RIGHT | GCAAGAGATGGTTGTGTTCCCTCAGACGACCCGAGCC | |
| ncov_cap_103_RIGHT | GCATGGCCTCTTATTGTAACAGCCAGACGACCCGAGCC | |
| ncov_cap_105_RIGHT | ATGGAACTGGTACTATCTATACAGAACTGCAGACGACCCGAGCC | |
| ncov_cap_107_RIGHT | ACACACACTGGTACTGGTCAGGCAGACGACCCGAGCC | |
| ncov_cap_109_RIGHT | AAACACAGTCTGTACCGTCTGCCAGACGACCCGAGCC | |
| ncov_cap_111_RIGHT | AGGACGAAGATGACAATTTAATTGATTCTTCAGACGACCCGAGCC | |
| ncov_cap_113_RIGHT | GCATTTTGATGAAGGTAATTGTGACACACAGACGACCCGAGCC | |
| ncov_cap_115_RIGHT | GGTATGATTTCGGTGATTTCATACAAACCCAGACGACCCGAGCC | |
| ncov_cap_117_RIGHT | TGTTAACTGTTTGGATGACAGATGCCAGACGACCCGAGCC | |
| ncov_cap_119_RIGHT | TGTATGCTGCTGACCCTGCTATCAGACGACCCGAGCC | |
| ncov_cap_121_RIGHT | CCAACAATGTGTGATATCAGACAACTACTCAGACGACCCGAGCC | |
| ncov_cap_123_RIGHT | CGTAATGTCATCCCTACTATAACTCAAATGCAGACGACCCGAGCC | |
| ncov_cap_125_RIGHT | TTATGGCCTCACTTGTTCTTGCTCAGACGACCCGAGCC | |
| ncov_cap_127_RIGHT | AATGCACTTTTATCTACTGATGGTAACAAACAGACGACCCGAGCC | |
| ncov_cap_129_RIGHT | GGCTAGCATAAAGAACTTTAAGTCAGTTCAGACGACCCGAGCC | |
| ncov_cap_131_RIGHT | GGTACACTTATGATTGAACGGTTCGCAGACGACCCGAGCC | |
| ncov_cap_133_RIGHT | ACACACCGCATACAGTCTTACAGCAGACGACCCGAGCC | |
| ncov_cap_135_RIGHT | TGTGCTAATGGACAAGTTTTTGGTTCAGACGACCCGAGCC | |
| ncov_cap_137_RIGHT | TGCTGTCTGACAGAGAATTACATCTTCAGACGACCCGAGCC | |
| ncov_cap_139_RIGHT | GCCATTAAGTGCACCTACACTAGTCAGACGACCCGAGCC | |
| ncov_cap_141_RIGHT | CGCTGTTGATGCACTATGTGAGACAGACGACCCGAGCC | |
| ncov_cap_143_RIGHT | CAATGCCAGATTACGTGCTAAGCCAGACGACCCGAGCC | |
| ncov_cap_145_RIGHT | AGGGTGTTATCACGCATGATGTCAGACGACCCGAGCC | |
| ncov_cap_147_RIGHT | CAGCTCACTCTTGTAATGTAAACAGATTCAGACGACCCGAGCC | |
| ncov_cap_149_RIGHT | TCAAAACTGAAGGTTTATGTGTTGACACAGACGACCCGAGCC | |
| ncov_cap_151_RIGHT | TACCTTTACAGCTAGGTTTTTCTACAGGCAGACGACCCGAGCC | |
| ncov_cap_153_RIGHT | GGCTTTGAGTTGACATCTATGAAGTATTTTCAGACGACCCGAGCC | |
| ncov_cap_155_RIGHT | AGCTAGTTGTGATGCAATCATGACTCAGACGACCCGAGCC | |
| ncov_cap_157_RIGHT | GTAGAATGGAAGTTCTATGATGCACAGCAGACGACCCGAGCC | |
| ncov_cap_159_RIGHT | CATTCCACACACCAGCTTTTGATAACAGACGACCCGAGCC | |
| ncov_cap_161_RIGHT | AGCTTGTGGGTTTACAAACAATTTGACAGACGACCCGAGCC | |
| ncov_cap_163_RIGHT | AATGTAGCATTTGAGCTTTGGGCCAGACGACCCGAGCC | |
| ncov_cap_165_RIGHT | AGAAATGCCCGTAATGGTGTTCTCAGACGACCCGAGCC | |
| ncov_cap_167_RIGHT | AGCTATGGATGAATTCATTGAACGGTACAGACGACCCGAGCC | |
| ncov_cap_169_RIGHT | CCCAAGATTTATCTGTAGTTTCTAAGGTTGCAGACGACCCGAGCC | |
| ncov_cap_171_RIGHT | GTCGCAAAATATACTCAACTGTGTCACAGACGACCCGAGCC | |
| ncov_cap_173_RIGHT | GATCTCATTATTAGTGATATGTACGACCCTCAGACGACCCGAGCC | |
| ncov_cap_175_RIGHT | AATTGGATGTAATTATCTTGGCAAACCACAGACGACCCGAGCC | |
| ncov_cap_177_RIGHT | TGATGTTCTTGTTAACAACTAAACGAACACAGACGACCCGAGCC | |
| ncov_cap_179_RIGHT | TGGGACCAATGGTACTAAGAGGTTCAGACGACCCGAGCC | |
| ncov_cap_181_RIGHT | CAAAAGTTGGATGGAAAGTGAGTTCACAGACGACCCGAGCC | |
| ncov_cap_183_RIGHT | ACTAGGTTTCAAACTTTACTTGCTTTACACAGACGACCCGAGCC | |
| ncov_cap_185_RIGHT | AGAGTCCAACCAACAGAATCTATTGTCAGACGACCCGAGCC | |
| ncov_cap_187_RIGHT | GCAGATTCATTTGTAATTAGAGGTGATGACAGACGACCCGAGCC | |
| ncov_cap_189_RIGHT | ATCTATCAGGCCGGTAGCACACCAGACGACCCGAGCC | |
| ncov_cap_191_RIGHT | ACAAAAAGTTTCTGCCTTTCCAACACAGACGACCCGAGCC | |
| ncov_cap_193_RIGHT | CAAACACGTGCAGGCTGTTTAACAGACGACCCGAGCC | |
| ncov_cap_195_RIGHT | TCTACCAGTGTCTATGACCAAGACACAGACGACCCGAGCC | |
| ncov_cap_197_RIGHT | CCATCAAAACCAAGCAAGAGGTCACAGACGACCCGAGCC | |
| ncov_cap_199_RIGHT | TCTGGTTGGACCTTTGGTGCAGCAGACGACCCGAGCC | |
| ncov_cap_201_RIGHT | ACGCTTGTTAAACAACTTAGCTCCCAGACGACCCGAGCC | |
| ncov_cap_203_RIGHT | TGGAAAGGGCTATCATCTTATGTCCTCAGACGACCCGAGCC | |
| ncov_cap_205_RIGHT | GTCTGGTAACTGTGATGTTGTAATAGGACAGACGACCCGAGCC | |
| ncov_cap_207_RIGHT | CTCATCGATCTCCAAGAACTTGGACAGACGACCCGAGCC | |
| ncov_cap_209_RIGHT | GGAACTGTAACTTTGAAGCAAGGTGCAGACGACCCGAGCC | |
| ncov_cap_211_RIGHT | AACAGTTTACTCACACCTTTTGCTCCAGACGACCCGAGCC | |
| ncov_cap_213_RIGHT | TCATTACTTCAGGTGATGGCACAACAGACGACCCGAGCC | |
| ncov_cap_215_RIGHT | CCAATTTATGATGAACCGACGACGCAGACGACCCGAGCC | |
| ncov_cap_217_RIGHT | AAAACCTTCTTTTTACGTTTACTCTCGTCAGACGACCCGAGCC | |
| ncov_cap_219_RIGHT | TGTCTTCTACAATTTGCCTATGCCACAGACGACCCGAGCC | |
| ncov_cap_221_RIGHT | CTTCTCAACGTGCCACTCCATGCAGACGACCCGAGCC | |
| ncov_cap_223_RIGHT | GTCGCTACAGGATTGGCAACTATCAGACGACCCGAGCC | |
| ncov_cap_225_RIGHT | TGAAGAGCAACCAATGGAGATTGACAGACGACCCGAGCC | |
| ncov_cap_227_RIGHT | TGTTCATCAGACAAGAGGAAGTTCACAGACGACCCGAGCC | |
| ncov_cap_229_RIGHT | TTTTCTTAGGAATCATCACAACTGTAGCCAGACGACCCGAGCC | |
| ncov_cap_231_RIGHT | TGCCAGGAACCTAAATTGGGTAGCAGACGACCCGAGCC | |
| ncov_cap_233_RIGHT | GGCCCCAAGGTTTACCCAATAATCAGACGACCCGAGCC | |
| ncov_cap_235_RIGHT | TGGACTTCCCTATGGTGCTAACAACAGACGACCCGAGCC | |
| ncov_cap_237_RIGHT | TTCAACTCCAGGCAGCAGTAGGCAGACGACCCGAGCC | |
| ncov_cap_239_RIGHT | GGTCCAGAACAAACCCAAGGAACAGACGACCCGAGCC | |
| ncov_cap_241_RIGHT | CGCATACAAAACATTCCCACCAACCAGACGACCCGAGCC | |
| ncov_cap_243_RIGHT | CGTTTTCGCTTTTCCGTTTACGACAGACGACCCGAGCC | |
| ncov_cap_245_RIGHT | TGGAAGAGCCCTAATGTGTAAAATTAATTTCAGACGACCCGAGCC | |
| RNAse_P_bead_rev | GTGGAGACAGCCGCTCACAGACGACCCGAGCC |
SARS-CoV-2 probe pool 2 [version 1]
| NAME | SEQUENCE | |
| ncov_cap_2_RIGHT | TCTTAAAGATGGCACTTGTGGCTTCAGACGACCCGAGCC | |
| ncov_cap_4_RIGHT | AGGAGCTGGTGGCCATAGTTACCAGACGACCCGAGCC | |
| ncov_cap_6_RIGHT | GCTTCATGCACTTTGTCCGAACCAGACGACCCGAGCC | |
| ncov_cap_8_RIGHT | TGATGGCTTTATGGGTAGAATTCGACAGACGACCCGAGCC | |
| ncov_cap_10_RIGHT | GCTGTTGTTAAAATTTATTGTCCAGCATCAGACGACCCGAGCC | |
| ncov_cap_12_RIGHT | TGGAGAAGGTTCCGAAGGTCTTCAGACGACCCGAGCC | |
| ncov_cap_14_RIGHT | GGAATATTGGTGAACAGAAATCAATACTGACAGACGACCCGAGCC | |
| ncov_cap_16_RIGHT | AGGTGGTGTTGTTCAGTTGACTCAGACGACCCGAGCC | |
| ncov_cap_18_RIGHT | TTTGGCTTTGTGTGCTGACTCTCAGACGACCCGAGCC | |
| ncov_cap_20_RIGHT | TTAGAACAACCTACTAGTGAAGCTGTTCAGACGACCCGAGCC | |
| ncov_cap_22_RIGHT | GCCTATACAGTTGAACTCGGTACAGCAGACGACCCGAGCC | |
| ncov_cap_24_RIGHT | TGTGAAGAAGAAGAGTTTGAGCCACAGACGACCCGAGCC | |
| ncov_cap_26_RIGHT | GGAACTTACACCAGTTGTTCAGACTCAGACGACCCGAGCC | |
| ncov_cap_28_RIGHT | CTTAAAGTGGGTGGTAGTTGTGTTTTCAGACGACCCGAGCC | |
| ncov_cap_30_RIGHT | CACAAATGTCTACTTAGCTGTCTTTGATCAGACGACCCGAGCC | |
| ncov_cap_32_RIGHT | CAATCTTCATCCAGATTCTGCCACTCAGACGACCCGAGCC | |
| ncov_cap_34_RIGHT | CAGTGCTTAAAAAGTGTAAAAGTGCCCAGACGACCCGAGCC | |
| ncov_cap_36_RIGHT | GTAAAACAACTGTAGCGTCACTTATCACAGACGACCCGAGCC | |
| ncov_cap_38_RIGHT | GTTCCTATAAAGATTGGTCCTATTCTGGACAGACGACCCGAGCC | |
| ncov_cap_40_RIGHT | ACTTATTTGGATGGAGCTGATGTTACTAACAGACGACCCGAGCC | |
| ncov_cap_42_RIGHT | CTTCTATTAAATGGGCAGATAACAACTGTCAGACGACCCGAGCC | |
| ncov_cap_44_RIGHT | AAGAGTCTTGAACGTGGTGTGTCAGACGACCCGAGCC | |
| ncov_cap_46_RIGHT | TGGTACATTTACTTGTGCTAGTGAGTACAGACGACCCGAGCC | |
| ncov_cap_48_RIGHT | AACCAACCATATCCAAACGCAAGCAGACGACCCGAGCC | |
| ncov_cap_50_RIGHT | CCTGGTGTATACGTTGTCTTTGGACAGACGACCCGAGCC | |
| ncov_cap_52_RIGHT | CACACAGATCTAATGGCTGCTTATGTCAGACGACCCGAGCC | |
| ncov_cap_54_RIGHT | GCAAAGAATACTGTTAAGAGTGTCGGTACAGACGACCCGAGCC | |
| ncov_cap_56_RIGHT | GTAGTGTTTGTCTTAGTGGTTTAGATTCTTCAGACGACCCGAGCC | |
| ncov_cap_58_RIGHT | ATGGCCCCGATTTCAGCTATGGCAGACGACCCGAGCC | |
| ncov_cap_60_RIGHT | CTGTGCTGGTAGTACATTTATTAGTGATGCAGACGACCCGAGCC | |
| ncov_cap_62_RIGHT | AGCTAATAACACTAAAGGTTCATTGCCTACAGACGACCCGAGCC | |
| ncov_cap_64_RIGHT | ACTCAAAACACTAGTTGCAACTGCCAGACGACCCGAGCC | |
| ncov_cap_66_RIGHT | GCTTGTATTGACTGTAGTGCGCCAGACGACCCGAGCC | |
| ncov_cap_68_RIGHT | AGCACTTAAGGGTGGTAAAATTGTTAATCAGACGACCCGAGCC | |
| ncov_cap_70_RIGHT | GCTGATTTTGACACATGGTTTAGCCCAGACGACCCGAGCC | |
| ncov_cap_72_RIGHT | GTGTTTTGGCTGCTGAATGTACACAGACGACCCGAGCC | |
| ncov_cap_74_RIGHT | GCTGGTGTTTGTGTATCTACTAGTGGCAGACGACCCGAGCC | |
| ncov_cap_76_RIGHT | GAGCTTTTGGTGAATACAGTCATGTAGCAGACGACCCGAGCC | |
| ncov_cap_78_RIGHT | TCCACAAAGCATTTCTATTGGTTCTTTCAGACGACCCGAGCC | |
| ncov_cap_80_RIGHT | GCTTGTTGTCATCTCGCAAAGGCAGACGACCCGAGCC | |
| ncov_cap_82_RIGHT | TGAAGACATGCTTAACCCTAATTATGAAGACAGACGACCCGAGCC | |
| ncov_cap_84_RIGHT | TGCTATGAGGCCCAATTTCACTCAGACGACCCGAGCC | |
| ncov_cap_86_RIGHT | TTTTAGCTTGGTTGTACGCTGCCAGACGACCCGAGCC | |
| ncov_cap_88_RIGHT | CCATATTGGGTAGTGCTTTATTAGAAGATGCAGACGACCCGAGCC | |
| ncov_cap_90_RIGHT | TGTCTGCTTTTGCAATGATGTTTGTCAGACGACCCGAGCC | |
| ncov_cap_92_RIGHT | ACTGTGTATGATGATGGTGCTAGGCAGACGACCCGAGCC | |
| ncov_cap_94_RIGHT | TGCCCTATTTTCTTCATAACTGGTAATACACAGACGACCCGAGCC | |
| ncov_cap_96_RIGHT | GGCAAACCTTGTATCAAAGTAGCCCAGACGACCCGAGCC | |
| ncov_cap_98_RIGHT | GCTTTGTGAAGAAATGCTGGACACAGACGACCCGAGCC | |
| ncov_cap_100_RIGHT | TGACCCAAATGTATAAACAGGCTAGACAGACGACCCGAGCC | |
| ncov_cap_102_RIGHT | AAATACGTGTGATGGTACAACATTTACTTCAGACGACCCGAGCC | |
| ncov_cap_104_RIGHT | ACAAACTGCTTGCACTGATGACACAGACGACCCGAGCC | |
| ncov_cap_106_RIGHT | CTTGGTAGTTTAGCTGCCACAGTCAGACGACCCGAGCC | |
| ncov_cap_108_RIGHT | TTTGGTGGTGCATCGTGTTGTCCAGACGACCCGAGCC | |
| ncov_cap_110_RIGHT | GTATACAGGGCTTTTGACATCTACAATGCAGACGACCCGAGCC | |
| ncov_cap_112_RIGHT | AATAGACGGTGACATGGTACCACCAGACGACCCGAGCC | |
| ncov_cap_114_RIGHT | CGCCAACTTAGGTGAACGTGTACAGACGACCCGAGCC | |
| ncov_cap_116_RIGHT | TGTTGACACTGACTTAACAAAGCCTTCAGACGACCCGAGCC | |
| ncov_cap_118_RIGHT | GTGAGAAAAATATTTGTTGATGGTGTTCCCAGACGACCCGAGCC | |
| ncov_cap_120_RIGHT | GCTTTTCAAACTGTCAAACCCGGCAGACGACCCGAGCC | |
| ncov_cap_122_RIGHT | CCTAGACAAATCAGCTGGTTTTCCACAGACGACCCGAGCC | |
| ncov_cap_124_RIGHT | TTGGAACAAGCAAATTCTATGGTGGCAGACGACCCGAGCC | |
| ncov_cap_126_RIGHT | TCACTATATGTTAAACCAGGTGGAACCCAGACGACCCGAGCC | |
| ncov_cap_128_RIGHT | AGACTTTGTGAATGAGTTTTACGCATCAGACGACCCGAGCC | |
| ncov_cap_130_RIGHT | CTCTCAACATACAATGCTAGTTAAACAGGCAGACGACCCGAGCC | |
| ncov_cap_132_RIGHT | AAGCTACATGATGAGTTAACAGGACACAGACGACCCGAGCC | |
| ncov_cap_134_RIGHT | TCTTGTCTGTTAATCCGTATGTTTGCCAGACGACCCGAGCC | |
| ncov_cap_136_RIGHT | TTTAGCTAACACCTGTACTGAAAGACTCAGACGACCCGAGCC | |
| ncov_cap_138_RIGHT | CAAATAGGAGAGTACACCTTTGAAAAAGGCAGACGACCCGAGCC | |
| ncov_cap_140_RIGHT | AAGGTTGGTATGCAAAAGTATTCTACACCAGACGACCCGAGCC | |
| ncov_cap_142_RIGHT | GTACTGTAAATGCATTGCCTGAGACCAGACGACCCGAGCC | |
| ncov_cap_144_RIGHT | TAGGTCCAGACATGTTCCTCGGCAGACGACCCGAGCC | |
| ncov_cap_146_RIGHT | GCCTCAAAGATTTTGGGACTACCACAGACGACCCGAGCC | |
| ncov_cap_148_RIGHT | CGTAGGAATGTGGCAACTTTACAAGCAGACGACCCGAGCC | |
| ncov_cap_150_RIGHT | CCCGCGAAGAAGCTATAAGACACAGACGACCCGAGCC | |
| ncov_cap_152_RIGHT | CCTCATACCACTTATGTACAAAGGACTCAGACGACCCGAGCC | |
| ncov_cap_154_RIGHT | GCATCATTCTATTGGATTTGATTACGTCTCAGACGACCCGAGCC | |
| ncov_cap_156_RIGHT | CTTGTAGAAAGGTTCAACACATGGTTCAGACGACCCGAGCC | |
| ncov_cap_158_RIGHT | TGCAATGTCGATAGATATCCTGCTAACAGACGACCCGAGCC | |
| ncov_cap_160_RIGHT | CCACTAAAGTCTGCTACGTGTATAACACAGACGACCCGAGCC | |
| ncov_cap_162_RIGHT | TGATGGACAACAGGGTGAAGTACCAGACGACCCGAGCC | |
| ncov_cap_164_RIGHT | CTCCAGCACATATATCTACTATTGGTGTCAGACGACCCGAGCC | |
| ncov_cap_166_RIGHT | AGAAGCCGTAAAAACACAGTTCAATTCAGACGACCCGAGCC | |
| ncov_cap_168_RIGHT | TGGTTTACATCTACTGATTGGACTAGCCAGACGACCCGAGCC | |
| ncov_cap_170_RIGHT | AATCTAGTCAAGCGTGGCAACCCAGACGACCCGAGCC | |
| ncov_cap_172_RIGHT | TTGCACCAGGTACAGCTGTTTTCAGACGACCCGAGCC | |
| ncov_cap_174_RIGHT | TGGAGGTTCCGTGGCTATAAAGATCAGACGACCCGAGCC | |
| ncov_cap_176_RIGHT | CAAATCCAATTCAGTTGTCTTCCTATTCTCAGACGACCCGAGCC | |
| ncov_cap_178_RIGHT | CAGTGTGTTAATCTTACAACCAGAACTCCAGACGACCCGAGCC | |
| ncov_cap_180_RIGHT | TTCGAAGACCCAGTCCCTACTTCAGACGACCCGAGCC | |
| ncov_cap_182_RIGHT | GCCTATTAATTTAGTGCGTGATCTCCCAGACGACCCGAGCC | |
| ncov_cap_184_RIGHT | TGAAAATGGAACCATTACAGATGCTGCAGACGACCCGAGCC | |
| ncov_cap_186_RIGHT | ACTGTGTTGCTGATTATTCTGTCCTCAGACGACCCGAGCC | |
| ncov_cap_188_RIGHT | CGTTATAGCTTGGAATTCTAACAATCTTGACAGACGACCCGAGCC | |
| ncov_cap_190_RIGHT | TTCTTTTGAACTTCTACATGCACCAGCAGACGACCCGAGCC | |
| ncov_cap_192_RIGHT | AACAAATACTTCTAACCAGGTTGCTGCAGACGACCCGAGCC | |
| ncov_cap_194_RIGHT | ACGTAGTGTAGCTAGTCAATCCATCACAGACGACCCGAGCC | |
| ncov_cap_196_RIGHT | ACTGGAATAGCTGTTGAACAAGACACAGACGACCCGAGCC | |
| ncov_cap_198_RIGHT | ATTGCTGCTAGAGACCTCATTTGTCAGACGACCCGAGCC | |
| ncov_cap_200_RIGHT | GAACCAAAAATTGATTGCCAACCAATCAGACGACCCGAGCC | |
| ncov_cap_202_RIGHT | AAGTTTGCAGACATATGTGACTCAACCAGACGACCCGAGCC | |
| ncov_cap_204_RIGHT | TGGAAAAGCACACTTTCCTCGTCAGACGACCCGAGCC | |
| ncov_cap_206_RIGHT | TTGATTTAGGTGACATCTCTGGCACAGACGACCCGAGCC | |
| ncov_cap_208_RIGHT | GCTGTATGACCAGTTGCTGTAGTCAGACGACCCGAGCC | |
| ncov_cap_210_RIGHT | CTTATTGTTGGCGTTGCACTTCTTCAGACGACCCGAGCC | |
| ncov_cap_212_RIGHT | GCCGTTCCAAAAACCCATTACTTCAGACGACCCGAGCC | |
| ncov_cap_214_RIGHT | TGTATTACACAGTTACTTCACTTCAGACTCAGACGACCCGAGCC | |
| ncov_cap_216_RIGHT | TTCTTGCTTTCGTGGTATTCTTGCCAGACGACCCGAGCC | |
| ncov_cap_218_RIGHT | CATGGCAGATTCCAACGGTACTCAGACGACCCGAGCC | |
| ncov_cap_220_RIGHT | GGATCACCGGTGGAATTGCTATCAGACGACCCGAGCC | |
| ncov_cap_222_RIGHT | ACGCTGTGACATCAAGGACCTGCAGACGACCCGAGCC | |
| ncov_cap_224_RIGHT | GTTGACTTTCAGGTTACTATAGCAGAGACAGACGACCCGAGCC | |
| ncov_cap_226_RIGHT | ATTCACCATTTCATCCTCTAGCTGATCAGACGACCCGAGCC | |
| ncov_cap_228_RIGHT | ATTTGTGCTTTTTAGCCTTTCTGCTACAGACGACCCGAGCC | |
| ncov_cap_230_RIGHT | CGTGTCCTATTCACTTCTATTCTAAATGGCAGACGACCCGAGCC | |
| ncov_cap_232_RIGHT | CCCAAAATCAGCGAAATGCACCCAGACGACCCGAGCC | |
| ncov_cap_234_RIGHT | GCAGTCCAGATGACCAAATTGGCCAGACGACCCGAGCC | |
| ncov_cap_236_RIGHT | CCTCAAGGAACAACATTGCCAAACAGACGACCCGAGCC | |
| ncov_cap_238_RIGHT | ACAACAACAAGGCCAAACTGTCCAGACGACCCGAGCC | |
| ncov_cap_240_RIGHT | ATTGGCATGGAAGTCACACCTTCAGACGACCCGAGCC | |
| ncov_cap_242_RIGHT | TTCTTCCTGCTGCAGATTTGGACAGACGACCCGAGCC | |
| ncov_cap_244_RIGHT | AGTGTGTAACATTAGGGAGGACTTGCAGACGACCCGAGCC | |
| RNAse_P_bead_rev | GTGGAGACAGCCGCTCACAGACGACCCGAGCC |
SARS-CoV-2 primer pool 1 [version 1]
| NAME | SEQUENCE | |
| ncov_cap_1_LEFT | ACGTGTGCTCTTCCGATCTTCTCTTGTAGATCTGTTCTCTAAACGAAC | |
| ncov_cap_3_LEFT | ACGTGTGCTCTTCCGATCTCTGTTTTACAGGTTCGCGACGT | |
| ncov_cap_5_LEFT | ACGTGTGCTCTTCCGATCTCCTCATGTGGGCGAAATACCAG | |
| ncov_cap_7_LEFT | ACGTGTGCTCTTCCGATCTATGGCTACCCTCTTGAGTGCAT | |
| ncov_cap_9_LEFT | ACGTGTGCTCTTCCGATCTGGGGAATGTCCAAATTTTGTATTTCCC | |
| ncov_cap_11_LEFT | ACGTGTGCTCTTCCGATCTCCACTTGCGAATTTTGTGGCAC | |
| ncov_cap_13_LEFT | ACGTGTGCTCTTCCGATCTCTATTGGGTTCCACGTGCTAGC | |
| ncov_cap_15_LEFT | ACGTGTGCTCTTCCGATCTTCAAACAAATTGTTGAATCCTGTGGT | |
| ncov_cap_17_LEFT | ACGTGTGCTCTTCCGATCTTGTTCACATCTGATTTGGCTACTAACA | |
| ncov_cap_19_LEFT | ACGTGTGCTCTTCCGATCTCCTGTGCAAAGGAAATTAAGGAGAGT | |
| ncov_cap_21_LEFT | ACGTGTGCTCTTCCGATCTTCCCACAGAAGTGTTAACAGAGGA | |
| ncov_cap_23_LEFT | ACGTGTGCTCTTCCGATCTAGTGCAAGGTTACAAGAGTGTGA | |
| ncov_cap_25_LEFT | ACGTGTGCTCTTCCGATCTGGCTTCACATATGTATTGTTCTTTCTACC | |
| ncov_cap_27_LEFT | ACGTGTGCTCTTCCGATCTAGGACAATCAGACAACTACTATTCAAACA | |
| ncov_cap_29_LEFT | ACGTGTGCTCTTCCGATCTGGCTACTAACAATGCCATGCAAG | |
| ncov_cap_31_LEFT | ACGTGTGCTCTTCCGATCTTAAGAGTTTGTGTAGATACTGTTCGCA | |
| ncov_cap_33_LEFT | ACGTGTGCTCTTCCGATCTTCTGGAAGAAACTAAGTTCCTCACAG | |
| ncov_cap_35_LEFT | ACGTGTGCTCTTCCGATCTTTATATAACCACTTACCCGGGTCAGG | |
| ncov_cap_37_LEFT | ACGTGTGCTCTTCCGATCTACAAGAGGGTGTGGTTGATTATGG | |
| ncov_cap_39_LEFT | ACGTGTGCTCTTCCGATCTACTTCTTCTTCTAAAACACCTGAAGAACA | |
| ncov_cap_41_LEFT | ACGTGTGCTCTTCCGATCTCGCAAGTTGTGGACATGTCAATG | |
| ncov_cap_43_LEFT | ACGTGTGCTCTTCCGATCTTGTCAGCATTAAATCACACTAAAAAGTGG | |
| ncov_cap_45_LEFT | ACGTGTGCTCTTCCGATCTAATGAGTTACTTGTTTCAACATGCCA | |
| ncov_cap_47_LEFT | ACGTGTGCTCTTCCGATCTACCTTTTGTTATGATGTCAGCACCA | |
| ncov_cap_49_LEFT | ACGTGTGCTCTTCCGATCTAGAAAGACAATTCTTATTTCACAGAGCAA | |
| ncov_cap_51_LEFT | ACGTGTGCTCTTCCGATCTTGTTTGGCATGTTAACAATGCAACT | |
| ncov_cap_53_LEFT | ACGTGTGCTCTTCCGATCTACCGAAGTTGTAGGAGACATTATACTTAA | |
| ncov_cap_55_LEFT | ACGTGTGCTCTTCCGATCTAATTCTAGAATTAAAGCATCTATGCCGAC | |
| ncov_cap_57_LEFT | ACGTGTGCTCTTCCGATCTTTGAACTCTACTAATGTCACTATTGCAACC | |
| ncov_cap_59_LEFT | ACGTGTGCTCTTCCGATCTGCAATTGTTTTTCAGCTATTTTGCAGT | |
| ncov_cap_61_LEFT | ACGTGTGCTCTTCCGATCTTGTCTATGCTAATGGAGGTAAAGGC | |
| ncov_cap_63_LEFT | ACGTGTGCTCTTCCGATCTAAGACTTATGAAAGACATTCTCTCTCTCA | |
| ncov_cap_65_LEFT | ACGTGTGCTCTTCCGATCTTAGTGCGGAAGTTGCAGTTAAAATG | |
| ncov_cap_67_LEFT | ACGTGTGCTCTTCCGATCTGCGATAGTTGTAATAACTATATGCTCACC | |
| ncov_cap_69_LEFT | ACGTGTGCTCTTCCGATCTACTTACCTTTTAAGTTGACATGTGCAA | |
| ncov_cap_71_LEFT | ACGTGTGCTCTTCCGATCTTCACTCGTGACATAGCATCTACAGA | |
| ncov_cap_73_LEFT | ACGTGTGCTCTTCCGATCTAACATCTGTTACACACCATCAAAACTT | |
| ncov_cap_75_LEFT | ACGTGTGCTCTTCCGATCTTTCTGAGTACTGTAGGCACGGC | |
| ncov_cap_77_LEFT | ACGTGTGCTCTTCCGATCTATGAGGTTTAGAAGAGCTTTTGGTGA | |
| ncov_cap_79_LEFT | ACGTGTGCTCTTCCGATCTTGTTCACACCTTTAGTACCTTTCTGG | |
| ncov_cap_81_LEFT | ACGTGTGCTCTTCCGATCTAGTACAAGTATTTTAGTGGAGCAATGGA | |
| ncov_cap_83_LEFT | ACGTGTGCTCTTCCGATCTAACGGTCTTTGGCTTGATGACG | |
| ncov_cap_85_LEFT | ACGTGTGCTCTTCCGATCTTAAGTTTGTTCGCATTCAACCAGGA | |
| ncov_cap_87_LEFT | ACGTGTGCTCTTCCGATCTTGGACCTTTTGTTGACAGGCAA | |
| ncov_cap_89_LEFT | ACGTGTGCTCTTCCGATCTGGAATTGCCGTTTTAGATATGTGTGC | |
| ncov_cap_91_LEFT | ACGTGTGCTCTTCCGATCTGTCCAGAGTACTCAATGGTCTTTGT | |
| ncov_cap_93_LEFT | ACGTGTGCTCTTCCGATCTACTGTGTTATGTATGCATCAGCTGT | |
| ncov_cap_95_LEFT | ACGTGTGCTCTTCCGATCTACAACTGTCATGTTTTTGGCCAG | |
| ncov_cap_97_LEFT | ACGTGTGCTCTTCCGATCTACTACTCCCACCCAAGAATAGCA | |
| ncov_cap_99_LEFT | ACGTGTGCTCTTCCGATCTTGGTTTCACTACTTTCTGTTTTGCTT | |
| ncov_cap_101_LEFT | ACGTGTGCTCTTCCGATCTAATTTGACCGTGATGCAGCCAT | |
| ncov_cap_103_LEFT | ACGTGTGCTCTTCCGATCTACCTCTTACAACAGCAGCCAAAC | |
| ncov_cap_105_LEFT | ACGTGTGCTCTTCCGATCTGCTTAGTCCTGTTGCACTACGAC | |
| ncov_cap_107_LEFT | ACGTGTGCTCTTCCGATCTGGTATGGTACTTGGTAGTTTAGCTGC | |
| ncov_cap_109_LEFT | ACGTGTGCTCTTCCGATCTTCAGGCAATAACAGTTACACCGG | |
| ncov_cap_111_LEFT | ACGTGTGCTCTTCCGATCTAGTCAGCTGATGCACAATCGTT | |
| ncov_cap_113_LEFT | ACGTGTGCTCTTCCGATCTACACTTTCTCTAACTACCAACATGAAGA | |
| ncov_cap_115_LEFT | ACGTGTGCTCTTCCGATCTAAGGACTGGTATGATTTTGTAGAAAACCC | |
| ncov_cap_117_LEFT | ACGTGTGCTCTTCCGATCTTGTTAATGCCTATATTAACCTTGACCAGG | |
| ncov_cap_119_LEFT | ACGTGTGCTCTTCCGATCTTGTTTTATTCTCTACAGTGTTCCCACC | |
| ncov_cap_121_LEFT | ACGTGTGCTCTTCCGATCTTGTTGCTTTTCAAACTGTCAAACCC | |
| ncov_cap_123_LEFT | ACGTGTGCTCTTCCGATCTGATAAGTACTTTGATTGTTACGATGGTGG | |
| ncov_cap_125_LEFT | ACGTGTGCTCTTCCGATCTTCAAAAATTATTGAAATCAATAGCCGCCA | |
| ncov_cap_127_LEFT | ACGTGTGCTCTTCCGATCTTGTAGCTTGTCACACCGTTTCT | |
| ncov_cap_129_LEFT | ACGTGTGCTCTTCCGATCTTGTCCGCAATTTACAACACAGACT | |
| ncov_cap_131_LEFT | ACGTGTGCTCTTCCGATCTATGTCTGAAGCAAAATGTTGGACTG | |
| ncov_cap_133_LEFT | ACGTGTGCTCTTCCGATCTATGCTTACCCACTTACTAAACATCCT | |
| ncov_cap_135_LEFT | ACGTGTGCTCTTCCGATCTGTTGTAAATGCTGTTACGACCATGT | |
| ncov_cap_137_LEFT | ACGTGTGCTCTTCCGATCTAGCGATAATGTTACTGACTTTAATGCAA | |
| ncov_cap_139_LEFT | ACGTGTGCTCTTCCGATCTCCACTTAACCGAAATTATGTCTTTACTGG | |
| ncov_cap_141_LEFT | ACGTGTGCTCTTCCGATCTACCCAACACTCAATATCTCAGATGAG | |
| ncov_cap_143_LEFT | ACGTGTGCTCTTCCGATCTAATGTAGTAGAATTATACCTGCACGTGC | |
| ncov_cap_145_LEFT | ACGTGTGCTCTTCCGATCTACCAGAATATTTCAATTCAGTGTGTAGAC | |
| ncov_cap_147_LEFT | ACGTGTGCTCTTCCGATCTGGTAAGAGAATTCCTTACACGTAACCC | |
| ncov_cap_149_LEFT | ACGTGTGCTCTTCCGATCTCTGATAGAGACCTTTATGACAAGTTGCA | |
| ncov_cap_151_LEFT | ACGTGTGCTCTTCCGATCTAGGACATGACCTATAGAAGACTCATCT | |
| ncov_cap_153_LEFT | ACGTGTGCTCTTCCGATCTTTTTCCAGAGTTAGTGCTAAACCACC | |
| ncov_cap_155_LEFT | ACGTGTGCTCTTCCGATCTTAGACGTGCCACATGCTTTTCC | |
| ncov_cap_157_LEFT | ACGTGTGCTCTTCCGATCTTGTTAAGCGTGTTGACTGGACT | |
| ncov_cap_159_LEFT | ACGTGTGCTCTTCCGATCTCTATTCTTATGCCACACATTCTGACAAA | |
| ncov_cap_161_LEFT | ACGTGTGCTCTTCCGATCTTCTGACAGTCCATGTGAGTCTCA | |
| ncov_cap_163_LEFT | ACGTGTGCTCTTCCGATCTTGGAACACTTTTACAAGACTTCAGAGT | |
| ncov_cap_165_LEFT | ACGTGTGCTCTTCCGATCTGGGTGTGGACATTGCTGCTAAT | |
| ncov_cap_167_LEFT | ACGTGTGCTCTTCCGATCTAACAAGCTAGTCTTAATGGAGTCACA | |
| ncov_cap_169_LEFT | ACGTGTGCTCTTCCGATCTTCTACTGATTGGACTAGCTAAACGTTT | |
| ncov_cap_171_LEFT | ACGTGTGCTCTTCCGATCTTGGTGTAAAGATGGCCATGTAGAA | |
| ncov_cap_173_LEFT | ACGTGTGCTCTTCCGATCTCATTTTGGTGCTGGTTCTGATAAAGG | |
| ncov_cap_175_LEFT | ACGTGTGCTCTTCCGATCTTTCACTTACATTTGTGGGTTTATACAACA | |
| ncov_cap_177_LEFT | ACGTGTGCTCTTCCGATCTACAAATCCAATTCAGTTGTCTTCCTATTC | |
| ncov_cap_179_LEFT | ACGTGTGCTCTTCCGATCTAGTCAGTGTGTTAATCTTACAACCAGA | |
| ncov_cap_181_LEFT | ACGTGTGCTCTTCCGATCTTTTGCTTCCACTGAGAAGTCTAACA | |
| ncov_cap_183_LEFT | ACGTGTGCTCTTCCGATCTACCTTGAAGGAAAACAGGGTAATTTCA | |
| ncov_cap_185_LEFT | ACGTGTGCTCTTCCGATCTTGCTGCAGCTTATTATGTGGGT | |
| ncov_cap_187_LEFT | ACGTGTGCTCTTCCGATCTAGTTTTTAACGCCACCAGATTTGC | |
| ncov_cap_189_LEFT | ACGTGTGCTCTTCCGATCTGGCAAACTGGAAAGATTGCTGAT | |
| ncov_cap_191_LEFT | ACGTGTGCTCTTCCGATCTACCCACTAATGGTGTTGGTTACC | |
| ncov_cap_193_LEFT | ACGTGTGCTCTTCCGATCTTCTTGACATTACACCATGTTCTTTTGG | |
| ncov_cap_195_LEFT | ACGTGTGCTCTTCCGATCTAGGTATATGCGCTAGTTATCAGACTCA | |
| ncov_cap_197_LEFT | ACGTGTGCTCTTCCGATCTCAATCTTTTGTTGCAATATGGCAGTTT | |
| ncov_cap_199_LEFT | ACGTGTGCTCTTCCGATCTAAGTGACACTTGCAGATGCTGG | |
| ncov_cap_201_LEFT | ACGTGTGCTCTTCCGATCTTGGCTTATAGGTTTAATGGTATTGGAGT | |
| ncov_cap_203_LEFT | ACGTGTGCTCTTCCGATCTACAAAGTTGAGGCTGAAGTGCA | |
| ncov_cap_205_LEFT | ACGTGTGCTCTTCCGATCTACTTATGTCCCTGCACAAGAAAAGA | |
| ncov_cap_207_LEFT | ACGTGTGCTCTTCCGATCTTGATCCTTTGCAACCTGAATTAGACT | |
| ncov_cap_209_LEFT | ACGTGTGCTCTTCCGATCTTGGTGACAATTATGCTTTGCTGTATG | |
| ncov_cap_211_LEFT | ACGTGTGCTCTTCCGATCTGTTCGCGCTACTGCAACGATAC | |
| ncov_cap_213_LEFT | ACGTGTGCTCTTCCGATCTATGCTTTAGTCTACTTCTTGCAGAGT | |
| ncov_cap_215_LEFT | ACGTGTGCTCTTCCGATCTACACAGTTACTTCACTTCAGACTATTACC | |
| ncov_cap_217_LEFT | ACGTGTGCTCTTCCGATCTACAAGCTGATGAGTACGAACTTATGT | |
| ncov_cap_219_LEFT | ACGTGTGCTCTTCCGATCTTCTAGAGTTCCTGATCTTCTGGTCT | |
| ncov_cap_221_LEFT | ACGTGTGCTCTTCCGATCTGGCCAGTAACTTTAGCTTGTTTTGT | |
| ncov_cap_223_LEFT | ACGTGTGCTCTTCCGATCTGTAATCGGAGCTGTGATCCTTCG | |
| ncov_cap_225_LEFT | ACGTGTGCTCTTCCGATCTTGCTTGTACAGTAAGTGACAACAGA | |
| ncov_cap_227_LEFT | ACGTGTGCTCTTCCGATCTACAACAGTACTTTTAAAAGAACCTTGCT | |
| ncov_cap_229_LEFT | ACGTGTGCTCTTCCGATCTTGCTTCACACTCAAAAGAAAGACAGA | |
| ncov_cap_231_LEFT | ACGTGTGCTCTTCCGATCTTGTAGTTGATGACCCGTGTCCT | |
| ncov_cap_233_LEFT | ACGTGTGCTCTTCCGATCTAGAGTATCATGACGTTCGTGTTGT | |
| ncov_cap_235_LEFT | ACGTGTGCTCTTCCGATCTAGGAAGACCTTAAATTCCCTCGAGG | |
| ncov_cap_237_LEFT | ACGTGTGCTCTTCCGATCTTGAGGGAGCCTTGAATACACCA | |
| ncov_cap_239_LEFT | ACGTGTGCTCTTCCGATCTCTCTTGCTTTGCTGCTGCTTGA | |
| ncov_cap_241_LEFT | ACGTGTGCTCTTCCGATCTCATTGGCCGCAAATTGCACAAT | |
| ncov_cap_243_LEFT | ACGTGTGCTCTTCCGATCTAGGCTGATGAAACTCAAGCCTT | |
| ncov_cap_245_LEFT | ACGTGTGCTCTTCCGATCTTGAATTCTCGTAACTACATAGCACAAGT | |
| RNAse_P_LEFT | ACGTGTGCTCTTCCGATCTGATTTGGACCTGCGAGCG |
SARS-CoV-2 primer pool 2 [version 1]
| NAME | SEQUENCE | |
| ncov_cap_2_LEFT | ACGTGTGCTCTTCCGATCTTCAGCACATCTAGGTTTCGTCC | |
| ncov_cap_4_LEFT | ACGTGTGCTCTTCCGATCTCATCAAACGTTCGGATGCTCGA | |
| ncov_cap_6_LEFT | ACGTGTGCTCTTCCGATCTTGGCACTGATCCTTATGAAGATTTTCA | |
| ncov_cap_8_LEFT | ACGTGTGCTCTTCCGATCTGCATGAAATTGCTTGGTACACGG | |
| ncov_cap_10_LEFT | ACGTGTGCTCTTCCGATCTGCGTCACCAAATGAATGCAACC | |
| ncov_cap_12_LEFT | ACGTGTGCTCTTCCGATCTACCTGAGCATAGTCTTGCCGAA | |
| ncov_cap_14_LEFT | ACGTGTGCTCTTCCGATCTGTGACTTTAAACTTAATGAAGAGATCGCC | |
| ncov_cap_16_LEFT | ACGTGTGCTCTTCCGATCTTGTACGATCAATTTTCTCCCGCA | |
| ncov_cap_18_LEFT | ACGTGTGCTCTTCCGATCTAAACCCGTCCTTGATTGGCTTG | |
| ncov_cap_20_LEFT | ACGTGTGCTCTTCCGATCTTTTGTCACGCACTCAAAGGGAT | |
| ncov_cap_22_LEFT | ACGTGTGCTCTTCCGATCTGCCCTTGCACCTAATATGATGGT | |
| ncov_cap_24_LEFT | ACGTGTGCTCTTCCGATCTTGCTGTCATAAAAACTTTGCAACCA | |
| ncov_cap_26_LEFT | ACGTGTGCTCTTCCGATCTGGTAAACCTTTGGAATTTGGTGCC | |
| ncov_cap_28_LEFT | ACGTGTGCTCTTCCGATCTACATTAAAAATGCAGACATTGTGGAAGA | |
| ncov_cap_30_LEFT | ACGTGTGCTCTTCCGATCTTGTCTTCATGTTGTCGGCCCAA | |
| ncov_cap_32_LEFT | ACGTGTGCTCTTCCGATCTAAGATCGCTGAGATTCCTAAAGAGG | |
| ncov_cap_34_LEFT | ACGTGTGCTCTTCCGATCTGTGGGTGATGTTGTTCAAGAGGG | |
| ncov_cap_36_LEFT | ACGTGTGCTCTTCCGATCTTGGAACTGTTTCTTGGAATTTGCG | |
| ncov_cap_38_LEFT | ACGTGTGCTCTTCCGATCTTGTAACACATGGCTTAAATTTGGAAGAA | |
| ncov_cap_40_LEFT | ACGTGTGCTCTTCCGATCTTCCTACCACATTCCACCTAGATGG | |
| ncov_cap_42_LEFT | ACGTGTGCTCTTCCGATCTAACCTCATAATTCACATGAAGGTAAAACA | |
| ncov_cap_44_LEFT | ACGTGTGCTCTTCCGATCTAGAGTTGAAGTTTAATCCACCTGCT | |
| ncov_cap_46_LEFT | ACGTGTGCTCTTCCGATCTCCTTAAGGGTGTAGAAGCTGTTATGT | |
| ncov_cap_48_LEFT | ACGTGTGCTCTTCCGATCTCCTCAGAATACAAAGGTCCTATTACGG | |
| ncov_cap_50_LEFT | ACGTGTGCTCTTCCGATCTAGAAACCTGCTTCAAGAGAGCTT | |
| ncov_cap_52_LEFT | ACGTGTGCTCTTCCGATCTGGGAATGGATAATCTTGCCTGCG | |
| ncov_cap_54_LEFT | ACGTGTGCTCTTCCGATCTTGCTAAGCCTTTTCTTAACAAAGTTGT | |
| ncov_cap_56_LEFT | ACGTGTGCTCTTCCGATCTTGGTTTTTACTATTAAGTGTTTGCCTAGGT | |
| ncov_cap_58_LEFT | ACGTGTGCTCTTCCGATCTAATGGGATTTAACTGCTTTTGGCTT | |
| ncov_cap_60_LEFT | ACGTGTGCTCTTCCGATCTGCATGTTGTAGACGGTTGTAATTCA | |
| ncov_cap_62_LEFT | ACGTGTGCTCTTCCGATCTGACTTGTCACTACAGTTTAAAAGACCAA | |
| ncov_cap_64_LEFT | ACGTGTGCTCTTCCGATCTAGAATCATCTGCAAAATCAGCGTCT | |
| ncov_cap_66_LEFT | ACGTGTGCTCTTCCGATCTCTACTTTTATTTCAGCAGCTCGGC | |
| ncov_cap_68_LEFT | ACGTGTGCTCTTCCGATCTGCGCAGGTAGCAAAAAGTCACA | |
| ncov_cap_70_LEFT | ACGTGTGCTCTTCCGATCTGCAGTTAATTAAAGTTACACTTGTGTTCC | |
| ncov_cap_72_LEFT | ACGTGTGCTCTTCCGATCTTGCTGCAGTCATAACAAGAGAAGT | |
| ncov_cap_74_LEFT | ACGTGTGCTCTTCCGATCTACCAATGTACTAGAAGGTTCTGTTGC | |
| ncov_cap_76_LEFT | ACGTGTGCTCTTCCGATCTGATCTTTACCAGGAGTTTTCTGTGGT | |
| ncov_cap_78_LEFT | ACGTGTGCTCTTCCGATCTACTGTACTCTGTTTAACACCAGTTTACT | |
| ncov_cap_80_LEFT | ACGTGTGCTCTTCCGATCTCCTTTAGTACTTTTGAAGAAGCTGCG | |
| ncov_cap_82_LEFT | ACGTGTGCTCTTCCGATCTCCACAAACCTCTATCACCTCAGC | |
| ncov_cap_84_LEFT | ACGTGTGCTCTTCCGATCTGGCTGGTAATGTTCAACTCAGGG | |
| ncov_cap_86_LEFT | ACGTGTGCTCTTCCGATCTTGGTTCATGTGGTAGTGTTGGTT | |
| ncov_cap_88_LEFT | ACGTGTGCTCTTCCGATCTACCACAACTCTTAATGACTTTAACCTTGT | |
| ncov_cap_90_LEFT | ACGTGTGCTCTTCCGATCTACAATGCTCAGGTGTTACTTTCCA | |
| ncov_cap_92_LEFT | ACGTGTGCTCTTCCGATCTTTACCTTCTCTTGCCACTGTAGCT | |
| ncov_cap_94_LEFT | ACGTGTGCTCTTCCGATCTACACTTATGAATGTCTTGACACTCGT | |
| ncov_cap_96_LEFT | ACGTGTGCTCTTCCGATCTTGTACTTGTTACTTTGGCCTCTTTTGT | |
| ncov_cap_98_LEFT | ACGTGTGCTCTTCCGATCTAGTCTTACTCTCAGTTTTGCAACAACT | |
| ncov_cap_100_LEFT | ACGTGTGCTCTTCCGATCTGTTCCCTTCCATCATATGCAGCT | |
| ncov_cap_102_LEFT | ACGTGTGCTCTTCCGATCTGGGCAAAAGTTACTAGTGCTATGCA | |
| ncov_cap_104_LEFT | ACGTGTGCTCTTCCGATCTCAGGTTGTAGATGCAGATAGTAAAATTGT | |
| ncov_cap_106_LEFT | ACGTGTGCTCTTCCGATCTTGCACTGTTATCCGATTTACAGGA | |
| ncov_cap_108_LEFT | ACGTGTGCTCTTCCGATCTAAGTGCCTGCCAATTCAACTGT | |
| ncov_cap_110_LEFT | ACGTGTGCTCTTCCGATCTAAAAACACAGTCTGTACCGTCTGC | |
| ncov_cap_112_LEFT | ACGTGTGCTCTTCCGATCTAATTCCTAAAAACTAATTGTTGTCGCTTCC | |
| ncov_cap_114_LEFT | ACGTGTGCTCTTCCGATCTTCAACGTCTTACTAAATACACAATGGCA | |
| ncov_cap_116_LEFT | ACGTGTGCTCTTCCGATCTCGAAATGCTGGTATTGTTGGTGT | |
| ncov_cap_118_LEFT | ACGTGTGCTCTTCCGATCTTGACTTCACGGAAGAGAGGTTAAAAC | |
| ncov_cap_120_LEFT | ACGTGTGCTCTTCCGATCTCCACTTCAGAGAGCTAGGTGTTG | |
| ncov_cap_122_LEFT | ACGTGTGCTCTTCCGATCTCACTTCTTCTTTGCTCAGGATGGT | |
| ncov_cap_124_LEFT | ACGTGTGCTCTTCCGATCTCGCATATACAAAACGTAATGTCATCCC | |
| ncov_cap_126_LEFT | ACGTGTGCTCTTCCGATCTATGGGTTGGGATTATCCTAAATGTGA | |
| ncov_cap_128_LEFT | ACGTGTGCTCTTCCGATCTTCAGGAGATGCCACAACTGCTT | |
| ncov_cap_130_LEFT | ACGTGTGCTCTTCCGATCTTACTCTCTGACGATGCTGTTGTG | |
| ncov_cap_132_LEFT | ACGTGTGCTCTTCCGATCTAGATCCATCAAGAATCCTAGGGGC | |
| ncov_cap_134_LEFT | ACGTGTGCTCTTCCGATCTGGAACCTGAGTTTTATGAGGCTATGT | |
| ncov_cap_136_LEFT | ACGTGTGCTCTTCCGATCTACTCAACTTTACTTAGGAGGTATGAGCT | |
| ncov_cap_138_LEFT | ACGTGTGCTCTTCCGATCTACGCTCAAAGCTACTGAGGAGA | |
| ncov_cap_140_LEFT | ACGTGTGCTCTTCCGATCTACCGAGGTACAACAACTTACAAATTAAAT | |
| ncov_cap_142_LEFT | ACGTGTGCTCTTCCGATCTCTCTACTACCCTTCTGCTCGCA | |
| ncov_cap_144_LEFT | ACGTGTGCTCTTCCGATCTCTTTGATGAAATTTCAATGGCCACAAA | |
| ncov_cap_146_LEFT | ACGTGTGCTCTTCCGATCTGCACATAAAGACAAATCAGCTCAATGC | |
| ncov_cap_148_LEFT | ACGTGTGCTCTTCCGATCTGTTGATTCATCACAGGGCTCAGA | |
| ncov_cap_150_LEFT | ACGTGTGCTCTTCCGATCTTGGGTTACATCCTACACAGGCA | |
| ncov_cap_152_LEFT | ACGTGTGCTCTTCCGATCTGTCGAGGGGTGTCATGCTACTA | |
| ncov_cap_154_LEFT | ACGTGTGCTCTTCCGATCTAGTGACACACTTAAAAATCTCTCTGACA | |
| ncov_cap_156_LEFT | ACGTGTGCTCTTCCGATCTCAGGTAACCTACAAAGCAACCATGA | |
| ncov_cap_158_LEFT | ACGTGTGCTCTTCCGATCTCCCAGTTCTTCACGACATTGGT | |
| ncov_cap_160_LEFT | ACGTGTGCTCTTCCGATCTAACTTGCCTGGTTGTGATGGTG | |
| ncov_cap_162_LEFT | ACGTGTGCTCTTCCGATCTTGTAGACATCATGCTAATGAGTACAGAT | |
| ncov_cap_164_LEFT | ACGTGTGCTCTTCCGATCTCACAAAAGTTGATGGTGTTGATGTAGA | |
| ncov_cap_166_LEFT | ACGTGTGCTCTTCCGATCTACTGAAACGATTTGTGCACCACT | |
| ncov_cap_168_LEFT | ACGTGTGCTCTTCCGATCTCCAACAATTACCTGAAACTTACTTTACTCA | |
| ncov_cap_170_LEFT | ACGTGTGCTCTTCCGATCTGGTTCATCTAAGTGTGTGTGTTCTGT | |
| ncov_cap_172_LEFT | ACGTGTGCTCTTCCGATCTAGAATGCTATTAGAAAAGTGTGACCTTCA | |
| ncov_cap_174_LEFT | ACGTGTGCTCTTCCGATCTTGATGCAGATTCAACTTTGATTGGTG | |
| ncov_cap_176_LEFT | ACGTGTGCTCTTCCGATCTGCTGATCTTTATAAGCTCATGGGACA | |
| ncov_cap_178_LEFT | ACGTGTGCTCTTCCGATCTAGGGGTACTGCTGTTATGTCTTTAAA | |
| ncov_cap_180_LEFT | ACGTGTGCTCTTCCGATCTAGGACTTGTTCTTACCTTTCTTTTCCA | |
| ncov_cap_182_LEFT | ACGTGTGCTCTTCCGATCTGTTGGATGGAAAGTGAGTTCAGAGT | |
| ncov_cap_184_LEFT | ACGTGTGCTCTTCCGATCTTCGGCTTTAGAACCATTGGTAGATT | |
| ncov_cap_186_LEFT | ACGTGTGCTCTTCCGATCTAGTGTACGTTGAAATCCTTCACTGT | |
| ncov_cap_188_LEFT | ACGTGTGCTCTTCCGATCTACTTTTAAGTGTTATGGAGTGTCTCCT | |
| ncov_cap_190_LEFT | ACGTGTGCTCTTCCGATCTAGATTGTTTAGGAAGTCTAATCTCAAACCT | |
| ncov_cap_192_LEFT | ACGTGTGCTCTTCCGATCTATGGTTTAACAGGCACAGGTGT | |
| ncov_cap_194_LEFT | ACGTGTGCTCTTCCGATCTATCAACTTACTCCTACTTGGCGTG | |
| ncov_cap_196_LEFT | ACGTGTGCTCTTCCGATCTAACTCTATTGCCATACCCACAAATTTT | |
| ncov_cap_198_LEFT | ACGTGTGCTCTTCCGATCTAGTCAAACAAATTTACAAAACACCACCA | |
| ncov_cap_200_LEFT | ACGTGTGCTCTTCCGATCTTTGCCACCTTTGCTCACAGATG | |
| ncov_cap_202_LEFT | ACGTGTGCTCTTCCGATCTGCACTTGGAAAACTTCAAGATGTGG | |
| ncov_cap_204_LEFT | ACGTGTGCTCTTCCGATCTTGCTGCTACTAAAATGTCAGAGTGT | |
| ncov_cap_206_LEFT | ACGTGTGCTCTTCCGATCTTGTAACACAAAGGAATTTTTATGAACCACA | |
| ncov_cap_208_LEFT | ACGTGTGCTCTTCCGATCTGTAAACATTCAAAAAGAAATTGACCGCC | |
| ncov_cap_210_LEFT | ACGTGTGCTCTTCCGATCTGCTCAAAGGAGTCAAATTACATTACACA | |
| ncov_cap_212_LEFT | ACGTGTGCTCTTCCGATCTCCAAGGGTGTTCACTTTGTTTGC | |
| ncov_cap_214_LEFT | ACGTGTGCTCTTCCGATCTGCCAACTATTTTCTTTGCTGGCA | |
| ncov_cap_216_LEFT | ACGTGTGCTCTTCCGATCTTTCACACAATCGACGGTTCATCC | |
| ncov_cap_218_LEFT | ACGTGTGCTCTTCCGATCTCCTTACTGCGCTTCGATTGTGT | |
| ncov_cap_220_LEFT | ACGTGTGCTCTTCCGATCTAAGCTCCTTGAACAATGGAACCT | |
| ncov_cap_222_LEFT | ACGTGTGCTCTTCCGATCTTTCTTTCAGACTGTTTGCGCGT | |
| ncov_cap_224_LEFT | ACGTGTGCTCTTCCGATCTAACGCTTTCTTATTACAAATTGGGAGC | |
| ncov_cap_226_LEFT | ACGTGTGCTCTTCCGATCTTCTCAATTAGATGAAGAGCAACCAATGG | |
| ncov_cap_228_LEFT | ACGTGTGCTCTTCCGATCTCGTCTATCAGTTACGTGCCAGAT | |
| ncov_cap_230_LEFT | ACGTGTGCTCTTCCGATCTATTATCTTTTGGTTCTCACTTGAACTGC | |
| ncov_cap_232_LEFT | ACGTGTGCTCTTCCGATCTACATCGATATCGGTAATTATACAGTTTCCT | |
| ncov_cap_234_LEFT | ACGTGTGCTCTTCCGATCTACCCTCAGATTCAACTGGCAGT | |
| ncov_cap_236_LEFT | ACGTGTGCTCTTCCGATCTAATGAAAGATCTCAGTCCAAGATGGT | |
| ncov_cap_238_LEFT | ACGTGTGCTCTTCCGATCTGCAGTCAAGCCTCTTCTCGTTC | |
| ncov_cap_240_LEFT | ACGTGTGCTCTTCCGATCTGCAAAAACGTACTGCCACTAAAGC | |
| ncov_cap_242_LEFT | ACGTGTGCTCTTCCGATCTACACAGGTGCCATCAAATTGGA | |
| ncov_cap_244_LEFT | ACGTGTGCTCTTCCGATCTTGCTGACTCAACTCAGGCCTAA | |
| RNAse_P_LEFT | ACGTGTGCTCTTCCGATCTGATTTGGACCTGCGAGCG |
Preparation of barcoded beads for RNA capture
Preparation of barcoded beads for RNA capture
Note
The first stage in our workflow is the preparation of bead-bound DNA probes that will facilitate subsequent viral RNA capture, cDNA synthesis and multiplex PCR to generate ~200 nt amplicons covering the entire SARS-CoV-2 genome. We use two distinct sets of probes for this, requiring two independent bead preparations for each sample to be processed. Ultimately, this enables library preparation to be performed in two parallel reactions, designed such that neighbouring amplicons do not overlap within the same library PCR. This strategy protects against preferential amplification of short overlap products, and has been successfully developed and employed elsewhere (Quick et al. [2017] Nat Protoc. 12: 1261; https://www.protocols.io/view/ncov-2019-sequencing-protocol-bbmuik6w/abstract). Throughout the protocol we use the generic terms “pool 1” and “pool 2” to refer to our two capture probe sets, as well as the corresponding primer sets needed for library amplification.
We envisage that capture beads could be prepared in bulk in advance and supplied to testing labs in 96-well or 384-well format. Each uniquely barcoded bead preparation could thus be used for multiple sample batches, provided each batch is sequenced independently. The protocol below is for a small-scale preparation of up to 10 uniquely barcoded bead sets, to be used in development and evaluation of a prototype. We also provide an alternative, lower-cost bead preparation method at the end of this page.
Wash 200 µL of streptavidin-coated Dynabeads™ MyOne™ C1 magnetic beads as per the manufacturer’s instructions:
Resuspend the Dynabeads™ magnetic beads in the vial (vortex for >00:00:30 ).
Transfer 200 µL of Dynabeads™ magnetic beads to a tube.
Add an equal volume of 1X B&W buffer and resuspend.
Place the tube on a magnet for 00:01:00 and discard the supernatant.
Remove the tube from the magnet and resuspend the washed magnetic beads in 200 µL of 1X B&W buffer.
Repeat steps 1.4–1.5 twice, for a total of 3 washes.
Pellet beads once more, and resuspend in 400 µL of 2X B&W buffer.
For each unique barcode, take an aliquot of the prepared beads and immobilise an oligonucleotide of the structure 5'-[ biotin ][ spacer ][ partial Illumina p5 adapter ][ sample barcode ][ generic stub ]-3’ onto the beads as follows:
Add 40 µL of 5 μM oligonucleotide ([Biotin]-barcoded bead tether) to 40 µL of washed beads.
Incubate for 00:15:00 at room temperature using gentle rotation.
Separate the biotinylated DNA coated beads with a magnet for 2–3 min.
Wash the coated beads 2–3 times with 100 µL 1X B&W buffer.
Resuspend in 120 µL of nuclease-free water.
For each barcoded sample, generate two distinct libraries of bead-bound SARS-CoV-2 probes by annealing separate oligonucleotide pools with the general structure 3'-[ generic stub complement ][ SARS-CoV-2 target ]-5’ and performing strand extension:
Mix the following components in 0.2 mL PCR tubes:
Component Pool 1 Pool 2
Bead-bound template (previous step) 60 µL 60 µL
5X PrimeSTAR GXL buffer 20 µL 20 µL
dNTP mixture (2.5 mM each) 8 µL 8 µL
SARS-CoV-2 probe pool 1 or 2 (10 μM) 10 µL 10 µL
PrimeSTAR GXL DNA polymerase 2 µL 2 µL
Incubate the reaction as follows:
Step Temperature Time Cycles
Denaturation 98 °C 00:00:10 1
Annealing 55 °C 00:00:15 1
Extension 68 °C 00:02:00 1
Hold 4 °C Indefinite 1
For each bead sample, dissociate and remove the non-biotinylated strand as follows:
Pellet the beads on a magnet and wash once in 50 µL 1X SSC.
Resuspend the beads in 20 µL of freshly prepared 0.15 M NaOH and incubate at room temperature for 00:10:00 .
Pellet the beads on a magnet and discard the supernatant containing the non-biotinylated strand.
Wash the beads once with 50 µL 0.1M NaOH, once with 50 µL of 1X B&W buffer and once with 50 µL TE buffer.
Resuspend beads in 50 µL TE buffer.
Note
The above steps are designed to produce two libraries of single-stranded, bead-bound SARS-CoV-2 probes for each sample barcode, with the general structure 5'-[ biotin ][ spacer ][ partial Illumina p5 adapter ][ sample barcode ][ generic stub ][ SARS-CoV-2 target ]-3’. Once the method has been suitably tested and is ready for deployment at scale, we envisage that these steps could be simplified substantially by synthesising the biotinylated probe sets in full for each sample barcode and immobilising these directly onto the streptavidin-coated beads.
Sample collection and preparation
Sample collection and preparation
Collect nasopharyngeal swabs in a 1.5 mL volume of transport medium.
Note
We are currently testing a range of media with the aim of identifying a formulation that optimally preserves viral RNA and supports hybridisation with our bead-bound probes in the following step. These include UTM, Lysis/binding buffer and a combination of the two (see Materials). Alternatively, kit-extracted RNA or synthetic control RNA can be used.
Quantify 10 µL of swab diluent using the Qubit RNA HS assay or similar.
RNA capture
RNA capture
For each sample to be tested, transfer 2 x 25 µL of unique barcoded beads (one for each probe set) into separate 0.2 mL PCR tubes.
Note
Alternatively, use multi-well PCR plates. For 384-well plates, sample volumes will need to be scaled down in subsequent steps.
Pellet the beads on a magnet and discard the supernatant.
Add 50 µL of swab diluent directly to each bead pellet (i.e. 2 x 50 µL per swab sample) and resuspend completely by pipette mixing. Alternatively, use purified or synthetic control RNA in a buffer suitable for probe hybridisation e.g. 1X SSC.
Place reactions in a preheated thermal cycler with a heated lid and incubate at 75 °C for 00:02:00 , then ramp to 25 °C at a rate of -0.1℃/s and proceed immediately to the next step.
Pellet the beads on a magnet and discard the supernatant.
Resuspend each reaction in 20 µL of Washing buffer A.
Combine all “pool 1” samples together into a fresh tube and repeat this process for “pool 2”.
Note
For large sample batches it should only be necessary to pool a small aliquot of each reaction. The combined samples will be used as input for cDNA synthesis. We are currently testing whether there is a limit to the amount of beads that can be tolerated in the reverse transcription reaction, as well as the subsequent library amplification.
cDNA synthesis
cDNA synthesis
Wash each pooled bead sample once with an equivalent volume of Washing buffer A and twice with the same volume of Washing buffer B, using a magnet to separate the beads from the solution between each washing step. After the final wash, leave the beads suspended in Washing buffer B at room temperature and proceed immediately to the next step.
Prepare a reverse transcriptase master mix by combining the following components from the SuperScript™ IV First-Strand Synthesis System:
Component Volume
5X SSIV buffer 20 µL
10 mM dNTP mix (10 mM each) 5 µL
100 mM DTT 5 µL
Ribonuclease inhibitor (40 U/μL) 5 µL
DEPC-treated water 60 µL
Pellet beads from step 14 on a magnet and discard the supernatant. Pulse spin tubes for 5 s, return to the magnet and discard any remaining liquid to ensure all the Washing buffer B has been removed (residual buffer may inhibit the reverse transcriptase reaction).
Perform one final wash in 25 µL of reverse transcriptase master mix. Again pellet beads on a magnet and discard the supernatant.
Resuspend each pooled bead sample in 19 µL of reverse transcriptase master mix and add 1 µL of SuperScript™ IV Reverse Transcriptase (200 U/μL). Mix well by pipetting and briefly centrifuge.
Incubate the reactions at 55 °C for 00:10:00 .
Inactivate by incubating at 80 °C for 00:10:00 .
Pellet beads by placing each tube on a magnet. Discard the supernatant and wash once in TE buffer.
Library amplification
Library amplification
Note
Here we generate amplicon libraries from each pooled cDNA sample by multiplex PCR. We also incorporate Illumina sequencing adapters and additional barcodes at this stage, producing sequence-ready samples that could potentially be combined with additional library pools for further scalability. Sequencing adapters are added over two sequential rounds of PCR, although we envisage that a single step protocol may also be feasible.
Pellet the “pool 1” and “pool 2” cDNA-coated bead samples from the previous step on a magnet, discard the supernatant and resuspend each in 28 µL of nuclease-free water.
Combine the first-round PCR components in separate 0.2 mL PCR tubes:
Component Pool 1 Pool 2
cDNA-coated beads (pool 1 or 2) 26.7 µL 26.7 µL
5X Q5 reaction buffer 10 µL 10 µL
10 mM dNTPs 1 µL 1 µL
Illumina P5 index primer (10 μM) 2.5 µL 2.5 µL
SARS-CoV-2 primer pool 1 or 2 (10 μM) 9.3 µL 9.3 µL
Q5 Hot Start High-Fidelity Polymerase 0.5 µL 0.5 µL
Note
The SARS-CoV-2 primer pools contain 124 (pool 1) and 123 (pool 2) individual primer sequences. The concentration of each primer in the final reaction is 0.015 μM, as recommended in previous studies (Quick et al. [2017] Nat Protoc. 12: 1261).
Mix well by pipetting and briefly centrifuge.
Incubate the first-round PCR reactions as follows:
Step Temperature Time Cycles
Heat activation 98 °C 00:00:30 1
Denaturation 98 °C 00:00:15 25-35
Annealing/extension 65 °C 00:05:00 25-35
Hold 4 °C Indefinite 1
For each reaction, pellet the cDNA-coated beads on a magnet and transfer the supernatant containing the amplified product to a new tube.
Clean up each reaction by performing a left side size selection with a 1.2X volume of SPRIselect beads as per the manufacturer’s instructions (Beckman Coulter). Elute the sample in 40 µL of nuclease-free water.
Quantify 1 µL of each purified sample using the Qubit dsDNA HS assay or similar.
Combine the second-round PCR components in separate 0.2 mL PCR tubes:
Component Pool 1 Pool 2
Purified template (previous step) 10 ng 10 ng
5X PrimeSTAR GXL buffer 10 µL 10 µL
dNTP mixture (2.5 mM each) 4 µL 4 µL
Illumina P5 flow cell primer (10 μM) 1.25 µL 1.25 µL
Illumina P7 index primer (10 μM) 1.25 µL 1.25 µL
PrimeSTAR GXL DNA polymerase 1 µL 1 µL
Nuclease-free water to 50 µL to 50 µL
Mix well by pipetting and briefly centrifuge.
Incubate the second-round PCR reactions as follows:
Step Temperature Time Cycles
Denaturation 98 °C 00:00:10 6
Annealing 60 °C 00:00:15 6
Extension 68 °C 00:01:00 6
Hold 4 °C Indefinite 1
Clean up each reaction by performing a left side size selection with a 0.8X volume of SPRIselect beads as per the manufacturer’s instructions (Beckman Coulter). Elute the sample in 40 µL of nuclease-free water.
Library quantitation and sequencing
Library quantitation and sequencing
Determine the concentration of the final “pool 1” and “pool 2” multiplexed libraries in ng/μL using the Qubit dsDNA HS assay or similar.
Assess the fragment size distribution of each library and determine the average fragment length using an Agilent 2100 Bioanalyzer or similar.
Calculate sample molarity using the following formula:
Concentration in nM = [(concentration in ng/μL) / (660 g/mol x average size in bp)] x 106
Combine an equimolar amount of each library and mix.
Note
If sequencing additional samples, quantitate them as above and combine at the desired relative molarity.
Sequence the final pooled library using an Illumina instrument, generating 2 x 150 nt paired end reads
Alternative bead preparation method
Alternative bead preparation method
Note
This alternative method enables the production of multiple uniquely barcoded bead preparations from a single biotinylated template oligo. This reduces oligo synthesis costs, but does introduce an additional level of protocol complexity.
Wash 200 µL of streptavidin-coated Dynabeads™ MyOne™ C1 magnetic beads as per the manufacturer’s instructions:
Resuspend the Dynabeads™ magnetic beads in the vial (vortex for >00:00:30 ).
Transfer 200 µL of Dynabeads™ magnetic beads to a tube.
Add an equal volume of 1X B&W buffer and resuspend.
Place the tube on a magnet for 00:01:00 and discard the supernatant.
Remove the tube from the magnet and resuspend the washed magnetic beads in 200 µL of 1X B&W buffer.
Repeat steps 38.4–38.5 twice, for a total of 3 washes.
Pellet beads once more, and resuspend in 400 µL of 2X B&W buffer.
Immobilise a generic oligonucleotide of the structure 5'-[ biotin ][ spacer ][ partial Illumina p5 adapter ]-3’ onto the beads as follows:
Add 400 µL of 5 μM oligonucleotide ([Biotin]-bead tether) to 400 µL of washed beads.
Incubate for 00:15:00 at room temperature using gentle rotation.
Separate the biotinylated DNA coated beads with a magnet for 2–3 min.
Wash the coated beads 2–3 times with 1X B&W buffer.
Resuspend in 1.200 mL of nuclease-free water.
For each unique barcode, generate two libraries of bead-bound SARS-CoV-2 probes via a two-stage annealing and extension process using a barcoded linker oligonucleotide of the structure 3'-[ partial Illumina p5 adapter complement ][ sample barcode ][ generic stub complement ]-5’ and two separate oligonucleotide pools with the general structure 3'-[ generic stub complement ][ SARS-CoV-2 target ]-5’:
Mix the following components in a 0.2 mL PCR tube:
Component Pool 1 Pool 2
Bead-bound template (previous step) 60 µL 60 µL
5X PrimeSTAR GXL buffer 20 µL 20 µL
dNTP mixture (2.5 mM each) 8 µL 8 µL
Barcoded linker (10 μM) 5 µL 5 µL
SARS-CoV-2 probe pool 1 or 2 (10 μM) 5 µL 5 µL
PrimeSTAR GXL DNA polymerase 2 µL 2 µL
Incubate the reaction as follows:
Step Temperature Time Cycles
Denaturation 98 °C 00:00:10 4
Annealing 55 °C 00:00:15 4
Extension 68 °C 00:02:00 4
Hold 4 °C Indefinite 4
For each bead sample, dissociate and remove the non-biotinylated strand as follows:
Pellet the beads on a magnet and wash once in 50 µL 1X SSC.
Resuspend the beads in 20 µL of freshly prepared 0.15 M NaOH and incubate at room temperature for 00:10:00 .
Pellet the beads on a magnet and discard the supernatant containing the non-biotinylated strand.
Wash the beads once with 50 µL 0.1M NaOH, once with 50 µL of 1X B&W buffer and once with 50 µL TE buffer.
Resuspend beads in 50 µL TE buffer.