TITLE: RTPCR Amplification of SARS-CoV2 Whole Genome for Illumina NGS 
AUTHORS: Monica Galiano, Shahjahan Miah, Angie Lackenby, Omolola Akinbami, Joanna Ellis, Maria Zambon

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><div class = "justify" style = "text-align:left">SUMMARY</div></div><div class = "text-block"><div class = "justify" style = "text-align:left">This document describes the procedure for performing RTPCR amplification for next-generation sequencing (NGS) of whole genomes from SARS-CoV2 positive clinical samples. This methodology was employed at the Respiratory Virus Unit, Microbiology Services Colindale, Public Health England (RVU-PHE) to sequence the first SARS-CoV2 positive samples.</div></div><div class = "text-block"><div class = "justify" style = "text-align:left">Reverse transcription (RT) is performed using random hexamer primers. PCR amplification is done using sequence-specific primers which amplify two sets of 30-31 overlapping amplicons (size 1.0 to 1.8 kb), each set independently covering the entire length of the MERS-CoV genome. Once amplicons are obtained, rough equimolar mixtures, clean-up, DNA quantitation and final dilutions are performed to prepare samples for library preparation for Illumina MiSeq next-generation sequencing (NGS). </div></div><div class = "text-block"><div class = "justify" style = "text-align:left">Primers from set A do not overlap location of primers from set B, except for those binding at the end of the genome (see figure 1). Amplicon mixtures from each set are treated as separate samples throughout the procedure, including assembly. Merging (or comparison) of consensus sequences of both sets is performed at the end. </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">IMPORTANT: Please note that every amplicon is obtained in a separate PCR reaction and, if using both primer sets, there is a total of ~61 PCR reactions; this protocol is NOT a multiplex approach. We recommend using a 96-well plate per sample.</div></div><div class = "text-block"><div class = "justify" style = "text-align:left"></div></div><div class = "text-block">ADVANTAGES AND LIMITATIONS</div><div class = "text-block">The use of both primer sets allows for redundancy in whole genome (WG) coverage, which increases the chances of obtaining full genome sequences when samples have low viral load. Although we haven’t formally determined the sensitivity of this assay (or limit of amplification of WG), we have obtained full coverage with samples with CT values of up to 38 (CT value obtained at RVU-PHE with RdRp gene detection within the SARS-CoV2 detection protocol as described by Corman et al. [1]). Variation in storage or transport conditions of samples may affect the integrity of viral nucleic acids.</div><div class = "text-block">Another advantage of this protocol is that, as primers from set A and B match different locations on the genome, direct comparison of the sequences obtained with each set allows for curation of primer-induced contamination of the sequences, although we deal with this with a specific step on the bioinformatic assembly pipeline (not described here).</div><div class = "text-block">Alternatively, when samples have a relatively good CT (<30) only set A may be used, simplifying the amplification and library steps.</div><div class = "text-block">The main limitation of this protocol is its unsuitability for high throughput processing of large batches of samples, although current efforts are being made to adapt the primers to a multiplex approach. </div></div>

[STEPS]
?. A total amount of 140 µl of RNA (includes some excess) are needed to set up the necessary volume of RT reactions. RNA should be kept on ice or stored at   if not immediately used.
-80 °C
?. To ensure uniform heat distribution, we aliquot this mix into 7 x 0.2 (strip) tubes.Cycling program: Hold at Keep cDNA on ice or store at  if not immediately used.
25 °C
42 °C
85 °C
4 °C
-80 °C
?. Prepare a PCR mix following Table  4. Thaw and briefly vortex all the required reagents except enzymes. Prepare a mix for ~70 samples (enough for both sets A and B).  ABC1PCR mixX  1 (µl)X  702  Water    35.8    2,506  310X High Fidelity Buffer (Invitrogen)  5.0    350  410 mM dNTPs mix (Invitrogen) See Appendix 1  1.0    70  5  50mM MgSO4 (Invitrogen)    2.0    140  6Platinum Taq DNA Polymerase 5 U/µl (Invitrogen)  0.2    14  7Final volume  44.0    3,080
Preparation of working dilution of dNTPs (100 mM dNTP Set, Invitrogen, cat 10297018)Mix the four tubes of dNTPs in one bijou tube. Add 1.5 ml of water to reach concentration of 10 mM (Final volume 2.5 ml). Aliquot in 2 ml tubes with screw cap.
ABC1PCR mixX  1 (µl)X  702  Water    35.8    2,506  310X High Fidelity Buffer (Invitrogen)  5.0    350  410 mM dNTPs mix (Invitrogen) See Appendix 1  1.0    70  5  50mM MgSO4 (Invitrogen)    2.0    140  6Platinum Taq DNA Polymerase 5 U/µl (Invitrogen)  0.2    14  7Final volume  44.0    3,080
?. Mix with a gentle vortex. Keep on ice. Dispense 44µl of the PCR mix into every well with primers of rows A, B, C, E, F and G.
?. Pull together all 7 cDNA strip tubes into one and load 4 µl of cDNA in each well.
?. Cover the 96-well plate with an adhesive lid or strip caps. Spin briefly the plate.
?. Transfer the 96-well plate to a thermal cycler. Amplify using the following cycling conditions:  followed by 35 cycles of:Hold at
95 °C
94 °C
52 °C
68 °C
15 °C
?. Working in a post-PCR area Use 1% or 2% E-gels 48 wells (Invitrogen) for visualisation of the bands, following manufacturer’s instructions. For each sample prepare 1x 96-well microtiter plate to mix Loading buffer
?. Prepare diluted Blue Juice loading buffer. Add 10 µl of diluted Blue Juice to every well of rows A, B, C, E, F and G (mirroring the PCR plate) Use multichannel pipettes.
Preparation of Blue Juice diluted 1/25 or 1/50Mix 10 ml of buffer TBE with 400 µl (1/25) or 200 µl (1/50) µl of undiluted Blue Juice.
?. Add  5 µl of sample from the PCR plate to the microtiter plate. Use multichannel pipettes.
?. Separately, prepare enough mixture of diluted Blue Juice (14µl diluted Blue Juice + 1µl of 1 Kb Plus Ladder) to add to wells marked with M.
?. Transfer 15 µl of samples and ladder dilutions into the E-gels. Load 15 µl of diluted Blue Juice in empty wells (no wells should be empty). Run for 20 minutes and view under shortwave U.V.
Alternative using multichannel pipette (tips will fit into every other well of 48 wells E-gels): if two samples are run in parallel, load amplicons from sample A in odd wells, and amplicons from sample 2 in even wells. Repeat the procedure with rows B to F. Load 15 µl of the mixture of Blue Juice + Ladder into the wells marked with M at the beginning and end of each gel row. In that way, amplicon 1 of sample A will run next to amplicon 1 of sample B and similarly for each amplicon.
?. To prepare equimolar mixes, ideally PCR reactions should be first cleaned up using e.g. silica columns or magnetic beads and then quantified, but the amount or PCR reactions makes this very impractical, unless using automated methods. Instead, rough equimolar mixes are prepared based on brightness of gel bands: 3 µl of a strong band, 5 µl of a normal band, and 10 µl of a weak or non-visible band. Mix the appropriate amount of each amplicon from SET A in an Eppendorf tube (final volume 60 to 300µl). Do not include the negative control. Repeat the procedure for SET B amplicons in a separate tube.
?. Perform PCR product purification using QIAquick PCR purification kit columns, following manufacturer’s instructions. Perform repeated additions and spins of the sample until all of it has gone through the column. Elute the purified DNA in 50 µl of molecular grade water.
?. Run quantitation of DNA using the Qubit dsDNA HS reagent (Life Technologies) and the Qubit fluorometer, according to manufacturer’s instructions.
?. Dilute the samples to the appropriate concentration for Illumina library preparation using Nextera XT (not described here).
?. The RT reaction is as follows (Table 1):  ABC1  RT mix  x 1 (µl)Bulk mix (x 14)2  Water    4.0    56  35X VILO  Reaction mix  4.0    56  4  10X Superscript Enzyme mix    2.0    28  5  RNA    10.0    140  6  Final volume    20.0    280
ABC1  RT mix  x 1 (µl)Bulk mix (x 14)2  Water    4.0    56  35X VILO  Reaction mix  4.0    56  4  10X Superscript Enzyme mix    2.0    28  5  RNA    10.0    140  6  Final volume    20.0    280
?. Combine primers according to Tables 1 and 2 (4.1) and dilute as follows to get a working primer dilution of 5 pmol/µlThaw both sets of primer mixes, briefly vortex them and spin down. Using a multichannel pipette (volume 0.5 or 1 to 10 µl) load 4 µl of primer mixes to the corresponding wells of a 96-well PCR plate, according to Figure 2. Keep the PCR plate on a cooler.
Preparation of primer mix pairs (working dilution 5 pmol/µl)Use different colours of strip tubes, e.g. yellow strip tubes for SET A and purple strip tubes for SET B. Add 180 µl of water + 10µl of each Forward and Reverse primers at 100 pmol/µl, according to Appendix 2. Vortex to mix. Store at -20°C.
?. The position of primers on the nCOV genome sequence has been based on the sequence of strain hCoV-19/Wuhan/IVDC-HB-01/2019 (GISAID accession EPI_ISL_402119). We gratefully acknowledge the authors (Wenjie Tan et al), originating and submitting laboratory  (National Institute for Viral Disease Control and Prevention, China CDC)  of this sequence from GISAID’s EpiCOV™ Database on which this protocol is based.   ABCDEFGH1Primer
  pairForward primerSequencePosition of
  5'endReverse primerSequencePosition of
  5'endAmplicon size21Ancov-1FATTAAAGGTTTATACCTTCCCAGGTAAC1ncov-1032R&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;TCAAAAGGTGTCTGCAATTCAT1032103132Ancov-15F&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;CCTTCCCAGGTAACAAACCA15ncov-1032R&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;TCAAAAGGTGTCTGCAATTCAT1032101743Ancov-693F&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;TTGACTTAGGCGACGAGCTT693ncov-2030R&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;CAGATGTGAACATCATAGCATCAA2030133754Ancov-1684F&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;CGCCATTATTTTGGCATCTT1684ncov-3019R&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;TGAAGCCAATTTAAACTCACCA3019133565Ancov-2665F&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;TGCCCTTGCACCTAATATGAT2665ncov-4010R&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;GGAACTTAGTTTCTTCCAGAGTTG4010134576Ancov-3686F&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;CACGAAGTTCTACTTGCACCA3686ncov-5015R&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;CAACTTGCGTGTGGAGGTTA5015132987Ancov-4667F&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;TCTCTCAAAGTGCCAGCTACAG4667ncov-6037R&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;GCTTGCGTTTGGATATGGTT6037137098Ancov-5687F&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;TCAGCACCACCTGCTCAGTA5687ncov-7021R&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;AACACCTAAAGCAGCGGTTG70211334109Ancov-6688F&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;TGCTAAGCCTTTTCTTAACAAAGTT6688ncov-8027R&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;CTGCAACTTCCGCACTATCA802713391110Ancov-7663F&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;TGATGAAGTTGCGAGAGACTTG7663ncov-9019R&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;TTCAGCAGCCAAAACACAAG901913561211Ancov-8700F&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;TTGATGGTGGTGTCACTCGT8700ncov-10033RAGAGGTTTGTGGTGGTTGGT1003313331312Ancov-9699F&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;TCATTTGTATTTCCACAAAGCA9699ncov-11071RCAAAGACCATTGAGTACTCTGGA1107113721413Ancov-10696FTGGAGACAGGTGGTTTCTCA10696ncov-12030RTGCATGGAAAGCAAAACAGA1203013341514Ancov-11668FCCGCTACTTTAGACTGACTCTTGG11668ncov-13050RGCAGGCACTTCTGTTGCAT1305013821615Ancov-12701FCCTGTTGCACTACGACAGATG12701ncov-14043RAATACCAGCATTTCGCATGG1404313421716Ancov-13663FCACACTTTCTCTAACTACCAACATGAA13663ncov-15028RATGCGAAAAGTGCATCTTGA1502813651817Ancov-14663FAAACTGTCAAACCCGGTAATTTT14663ncov-16023RAGACACGAACCGTTCAATCA1602313601918Ancov-15686FTGCGTAAACATTTCTCAATGATG15686ncov-17038RTTGCAACATTGCTAGAAAACTCA1703813522019Ancov-16689FTGCTACTGTACGTGAAGTGCTG16689ncov-18034RAAGTTGCCACATTCCTACGTG1803413452120Ancov-17694FTGCAATTAACAGGCCACAAA17694ncov-19031RTCGTGAAGAACTGGGAATTTG1903113372221Ancov-18683FCATGCTTTTCCACTGCTTCA18683ncov-20043RAACACCATTACGGGCATTTC2004313602322Ancov-19664FTTGATGGACAACAGGGTGAA19664ncov-21231RGTCCACCATGCGAAGTGTC2123115672423Ancov-20863FCATTTTGGTGCTGGTTCTGA20863ncov-22226RCGAAAAACCCTGAGGGAGAT2222613632524Ancov-21896FTTCGAAGACCCAGTCCCTAC21896ncov-23214RCACCTGTGCCTGTTAAACCA2321413182625Ancov-22883FTCTTGATTCTAAGGTTGGTGGT22883ncov-24231RCACCAAAGGTCCAACCAGAA2423113482726Ancov-23850FTTAAACCGTGCTTTAACTGGAATA23850ncov-25243RATGGCAATCAAGCCAGCTAT2524313932827Ancov-24858FGGCACACACTGGTTTGTAACAC24858ncov-26224RTGCTTACAAAGGCACGCTAGT2622413662928Ancov-25886FTCTTCAATTGTCATTACTTCAGGTG25886ncov-27227RCCTGAAAGTCAACGAGATGAAA2722713413029Ancov-26889FGCCACTCCATGGCACTATTC26889ncov-28191RTTCATAGAACGAACAACGCACT2819113023130Ancov-27876FTTGTCACGCCTAAACGAACA27876ncov-29226RACATTCCGAAGAACGCTGAA2922613503231Ancov-28871FAGGCAGCAGTAGGGGAACTT28871ncov-29848RAAAATCACATGGGGATAGCA29848977  ABCDEFGH1Primer
  pairForward primerSequencePosition of
  5'endReverse primerSequencePosition of
  5'endAmplicon size21Bncov-193F&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;CTTACGGTTTCGTCCGTGTT193ncov-1541R&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;GTGGAACCCAATAGGCACAC1541134832Bncov-1185F&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;CGTCACCAAATGAATGCAAC1185ncov-2495R&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;TTCTGTGGGAAGTGTTTCTCC2495131043Bncov-2193F&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;GAGACGGTTGGGAAATTGTT2193ncov-3523R&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;TTCAACTTGCATGGCATTGT3523133054Bncov-3172F&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;TCAACCTGAAGAAGAGCAAGAA3172ncov-4525R&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;AGCACCATAATCAACCACACC4525135365Bncov-4173F&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;CTAAAAAGGCTGGTGGCACT4173ncov-5550R&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;AGGGTTGTCTGCTGTTGTCC5550137776Bncov-5192F&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;CTGGGTAGGTACATGTCAGCA5192ncov-6548R&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;GATCTGTGTGGCCAACCTCT6548135687Bncov-6192F&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;CACCCTCTTTTAAGAAAGGAGCTA6192ncov-7537R&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;TGTACATTCGACTCTTGTTGCTC7537134598Bncov-7194F&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;CCATTTCATCTTTTAAATGGGATT7194ncov-8554R&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;CCACCCTTAAGTGCTATCTTTG85541360109Bncov-8179F&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;GCAAGGGTTTGTTGATTCAGA8179ncov-9506R&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;TAAAGGCAACTACATGACTGTATTCAC950613271110Bncov-9169F&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;CCTTGAAGGTTCTGTTAGAGTGG9169ncov-10456RGAAATTGGGCCTCATAGCAC1045612871211Bncov-10080FCATCTGGTAAAGTTGAGGGTTGT10080ncov-11533RTCTGGCCAAAAACATGACAG1153314531312Bncov-11189FCCTTCTCTTGCCACTGTAGCTT11189ncov-12547RTGCTGATGCATAAGTAAATGTTG1254713581413Bncov-12163FGGCTGTTGCTAATGGTGATTC12163ncov-13547RTCAAAAGCCCTGTATACGACA1354713841514Bncov-13191FCAGTTACACCGGAAGCCAAT13191ncov-14513RTCCTGATTATGTACAACACCTAGCTC1451313221615Bncov-14183FCCAGGGCTTTAACTGCAGAG14183ncov-15544RCCGTGACAGCTTGACAAATG1554413611716Bncov-15167FTGAAATCAATAGCCGCCACT15167ncov-16511RAAACCAAAAACTTGTCCATTAGC1651113441817Bncov-16176FTTCAAGGTATTGGGAACCTGA16176ncov-17551RCGAGGAACATGTCTGGACCTA1755113751918Bncov-17171FCCGCTGTTGATGCACTATGT17171ncov-18533RTTTATACGCACTACATTCCAAGG1853313622019Bncov-18165FACCTGGCATACCTAAGGACA18165ncov-19542RCATCATGTTATAAGCATCGAGATACA1954213772120Bncov-19191FTTGGAATTGCAATGTCGATAG19191ncov-20478RACCTGTTTGCGCATCTGTTA2047812872221Bncov-19966FTGTGCACCACTCACTGTCTT19966ncov-21730RAAGAACAAGTCCTGAGTTGAATG2173017642322Bncov-21360FCAAATCCAATTCAGTTGTCTTCC21360ncov-22705RCCATAACACTTAAAAGTGGAAAATGA2270513452423Bncov-22363FTGGGTTATCTTCAACCTAGGACTT22363ncov-23713RTTTGTGGGTATGGCAATAGAGTT2371313502524Bncov-23384FGGTTGCTGTTCTTTATCAGGATG23384ncov-24735RGAGGTGCTGACTGAGGGAAG2473513512625Bncov-24369FGACTCACTTTCTTCCACAGCAA24369ncov-25712RAGAGAAAAGGGGCTTCAAGG2571213432726Bncov-25354FTGCTCAAAGGAGTCAAATTACATTA25354ncov-26734RAAACAGCAGCAAGCACAAAA2673413802827Bncov-26361FTGTGTGCGTACTGCTGCAA26361ncov-27712RTGCCGCAACAATAAGAAAAA2771213512928Bncov-27361FTGAAGAGCAACCAATGGAGA27361ncov-28719RGGGTGCCAATGTGATCTTTT2871913583029Bncov-28397FGCCCCAAGGTTTACCCAATA28397ncov-29734RGGTGAAAATGTGGTGGCTCT2973413373130Bncov-28397FGCCCCAAGGTTTACCCAATA28397ncov-29871RTGATTTTAATAGCTTCTTAGGAGAATGAC298711474
ABCDEFGH1Primer
  pairForward primerSequencePosition of
  5'endReverse primerSequencePosition of
  5'endAmplicon size21Ancov-1FATTAAAGGTTTATACCTTCCCAGGTAAC1ncov-1032R&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;TCAAAAGGTGTCTGCAATTCAT1032103132Ancov-15F&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;CCTTCCCAGGTAACAAACCA15ncov-1032R&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;TCAAAAGGTGTCTGCAATTCAT1032101743Ancov-693F&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;TTGACTTAGGCGACGAGCTT693ncov-2030R&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;CAGATGTGAACATCATAGCATCAA2030133754Ancov-1684F&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;CGCCATTATTTTGGCATCTT1684ncov-3019R&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;TGAAGCCAATTTAAACTCACCA3019133565Ancov-2665F&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;TGCCCTTGCACCTAATATGAT2665ncov-4010R&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;GGAACTTAGTTTCTTCCAGAGTTG4010134576Ancov-3686F&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;CACGAAGTTCTACTTGCACCA3686ncov-5015R&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;CAACTTGCGTGTGGAGGTTA5015132987Ancov-4667F&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;TCTCTCAAAGTGCCAGCTACAG4667ncov-6037R&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;GCTTGCGTTTGGATATGGTT6037137098Ancov-5687F&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;TCAGCACCACCTGCTCAGTA5687ncov-7021R&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;AACACCTAAAGCAGCGGTTG70211334109Ancov-6688F&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;TGCTAAGCCTTTTCTTAACAAAGTT6688ncov-8027R&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;CTGCAACTTCCGCACTATCA802713391110Ancov-7663F&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;TGATGAAGTTGCGAGAGACTTG7663ncov-9019R&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;TTCAGCAGCCAAAACACAAG901913561211Ancov-8700F&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;TTGATGGTGGTGTCACTCGT8700ncov-10033RAGAGGTTTGTGGTGGTTGGT1003313331312Ancov-9699F&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;TCATTTGTATTTCCACAAAGCA9699ncov-11071RCAAAGACCATTGAGTACTCTGGA1107113721413Ancov-10696FTGGAGACAGGTGGTTTCTCA10696ncov-12030RTGCATGGAAAGCAAAACAGA1203013341514Ancov-11668FCCGCTACTTTAGACTGACTCTTGG11668ncov-13050RGCAGGCACTTCTGTTGCAT1305013821615Ancov-12701FCCTGTTGCACTACGACAGATG12701ncov-14043RAATACCAGCATTTCGCATGG1404313421716Ancov-13663FCACACTTTCTCTAACTACCAACATGAA13663ncov-15028RATGCGAAAAGTGCATCTTGA1502813651817Ancov-14663FAAACTGTCAAACCCGGTAATTTT14663ncov-16023RAGACACGAACCGTTCAATCA1602313601918Ancov-15686FTGCGTAAACATTTCTCAATGATG15686ncov-17038RTTGCAACATTGCTAGAAAACTCA1703813522019Ancov-16689FTGCTACTGTACGTGAAGTGCTG16689ncov-18034RAAGTTGCCACATTCCTACGTG1803413452120Ancov-17694FTGCAATTAACAGGCCACAAA17694ncov-19031RTCGTGAAGAACTGGGAATTTG1903113372221Ancov-18683FCATGCTTTTCCACTGCTTCA18683ncov-20043RAACACCATTACGGGCATTTC2004313602322Ancov-19664FTTGATGGACAACAGGGTGAA19664ncov-21231RGTCCACCATGCGAAGTGTC2123115672423Ancov-20863FCATTTTGGTGCTGGTTCTGA20863ncov-22226RCGAAAAACCCTGAGGGAGAT2222613632524Ancov-21896FTTCGAAGACCCAGTCCCTAC21896ncov-23214RCACCTGTGCCTGTTAAACCA2321413182625Ancov-22883FTCTTGATTCTAAGGTTGGTGGT22883ncov-24231RCACCAAAGGTCCAACCAGAA2423113482726Ancov-23850FTTAAACCGTGCTTTAACTGGAATA23850ncov-25243RATGGCAATCAAGCCAGCTAT2524313932827Ancov-24858FGGCACACACTGGTTTGTAACAC24858ncov-26224RTGCTTACAAAGGCACGCTAGT2622413662928Ancov-25886FTCTTCAATTGTCATTACTTCAGGTG25886ncov-27227RCCTGAAAGTCAACGAGATGAAA2722713413029Ancov-26889FGCCACTCCATGGCACTATTC26889ncov-28191RTTCATAGAACGAACAACGCACT2819113023130Ancov-27876FTTGTCACGCCTAAACGAACA27876ncov-29226RACATTCCGAAGAACGCTGAA2922613503231Ancov-28871FAGGCAGCAGTAGGGGAACTT28871ncov-29848RAAAATCACATGGGGATAGCA29848977
ABCDEFGH1Primer
  pairForward primerSequencePosition of
  5'endReverse primerSequencePosition of
  5'endAmplicon size21Bncov-193F&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;CTTACGGTTTCGTCCGTGTT193ncov-1541R&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;GTGGAACCCAATAGGCACAC1541134832Bncov-1185F&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;CGTCACCAAATGAATGCAAC1185ncov-2495R&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;TTCTGTGGGAAGTGTTTCTCC2495131043Bncov-2193F&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;GAGACGGTTGGGAAATTGTT2193ncov-3523R&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;TTCAACTTGCATGGCATTGT3523133054Bncov-3172F&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;TCAACCTGAAGAAGAGCAAGAA3172ncov-4525R&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;AGCACCATAATCAACCACACC4525135365Bncov-4173F&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;CTAAAAAGGCTGGTGGCACT4173ncov-5550R&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;AGGGTTGTCTGCTGTTGTCC5550137776Bncov-5192F&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;CTGGGTAGGTACATGTCAGCA5192ncov-6548R&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;GATCTGTGTGGCCAACCTCT6548135687Bncov-6192F&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;CACCCTCTTTTAAGAAAGGAGCTA6192ncov-7537R&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;TGTACATTCGACTCTTGTTGCTC7537134598Bncov-7194F&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;CCATTTCATCTTTTAAATGGGATT7194ncov-8554R&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;CCACCCTTAAGTGCTATCTTTG85541360109Bncov-8179F&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;GCAAGGGTTTGTTGATTCAGA8179ncov-9506R&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;TAAAGGCAACTACATGACTGTATTCAC950613271110Bncov-9169F&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;CCTTGAAGGTTCTGTTAGAGTGG9169ncov-10456RGAAATTGGGCCTCATAGCAC1045612871211Bncov-10080FCATCTGGTAAAGTTGAGGGTTGT10080ncov-11533RTCTGGCCAAAAACATGACAG1153314531312Bncov-11189FCCTTCTCTTGCCACTGTAGCTT11189ncov-12547RTGCTGATGCATAAGTAAATGTTG1254713581413Bncov-12163FGGCTGTTGCTAATGGTGATTC12163ncov-13547RTCAAAAGCCCTGTATACGACA1354713841514Bncov-13191FCAGTTACACCGGAAGCCAAT13191ncov-14513RTCCTGATTATGTACAACACCTAGCTC1451313221615Bncov-14183FCCAGGGCTTTAACTGCAGAG14183ncov-15544RCCGTGACAGCTTGACAAATG1554413611716Bncov-15167FTGAAATCAATAGCCGCCACT15167ncov-16511RAAACCAAAAACTTGTCCATTAGC1651113441817Bncov-16176FTTCAAGGTATTGGGAACCTGA16176ncov-17551RCGAGGAACATGTCTGGACCTA1755113751918Bncov-17171FCCGCTGTTGATGCACTATGT17171ncov-18533RTTTATACGCACTACATTCCAAGG1853313622019Bncov-18165FACCTGGCATACCTAAGGACA18165ncov-19542RCATCATGTTATAAGCATCGAGATACA1954213772120Bncov-19191FTTGGAATTGCAATGTCGATAG19191ncov-20478RACCTGTTTGCGCATCTGTTA2047812872221Bncov-19966FTGTGCACCACTCACTGTCTT19966ncov-21730RAAGAACAAGTCCTGAGTTGAATG2173017642322Bncov-21360FCAAATCCAATTCAGTTGTCTTCC21360ncov-22705RCCATAACACTTAAAAGTGGAAAATGA2270513452423Bncov-22363FTGGGTTATCTTCAACCTAGGACTT22363ncov-23713RTTTGTGGGTATGGCAATAGAGTT2371313502524Bncov-23384FGGTTGCTGTTCTTTATCAGGATG23384ncov-24735RGAGGTGCTGACTGAGGGAAG2473513512625Bncov-24369FGACTCACTTTCTTCCACAGCAA24369ncov-25712RAGAGAAAAGGGGCTTCAAGG2571213432726Bncov-25354FTGCTCAAAGGAGTCAAATTACATTA25354ncov-26734RAAACAGCAGCAAGCACAAAA2673413802827Bncov-26361FTGTGTGCGTACTGCTGCAA26361ncov-27712RTGCCGCAACAATAAGAAAAA2771213512928Bncov-27361FTGAAGAGCAACCAATGGAGA27361ncov-28719RGGGTGCCAATGTGATCTTTT2871913583029Bncov-28397FGCCCCAAGGTTTACCCAATA28397ncov-29734RGGTGAAAATGTGGTGGCTCT2973413373130Bncov-28397FGCCCCAAGGTTTACCCAATA28397ncov-29871RTGATTTTAATAGCTTCTTAGGAGAATGAC298711474
?. Some examples of gel pictures are shown here: