TITLE: INSIGHT: a population scale COVID-19 testing strategy combining point-of-care diagnosis with centralised high-throughput sequencing
AUTHORS: Qianxin  Wu, Chenqu Suo, Tom  Brown, Tengyao  Wang, Sarah Teichmann, Andrew Bassett

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">We present INSIGHT (Isothermal NASBA-Sequencing-based hIGH-througput Test): a two-stage COVID-19 testing strategy, using a barcoded isothermal NASBA reaction that combines point-of- care diagnosis with next generation sequencing, aiming to achieve population-scale COVID-19 testing. INSIGHT combines the advantages of near-patient with centralised testing. Stage 1 allows a quick decentralised readout for early isolation of pre-symptomatic or asymptomatic patients. The same reaction products can then be used in a highly multiplexed sequencing-based assay in Stage 2, confirming the near-patient testing results and facilitating centralised data collection. Based on experiments using commercially acquired human saliva with spiked-in viral RNA as input, the INSIGHT platform gives Stage 1 results within one to two hours, using either fluorescence detection or a lateral flow (dipstick) readout, whilst simultaneously incorporating sample-specific barcodes into the amplification product. INSIGHT Stage 2 can be performed by directly pooling and sequencing all post-amplification barcoded Stage 1 products from hundreds of thousands of samples with minimal sample preparation steps. The 95% limit of detection (LoD-95) for INSIGHT is estimated to be below 50 copies of viral RNA per 20 μl of reaction. Our two-stage testing strategy is suitable for further development into a rapid home-based and point-of-care assay, and is potentially scalable to the population level. 


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[STEPS]
?. Lysis of saliva samples
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							Mix crude saliva (commercial pooled human saliva from healthy individuals) at 1:1 ratio with QuickExtract DNA Extraction Solution. Incubate at 95 °C for 5 min to ensure complete lysis of virus and inactivation of proteinase K. 
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?. (Option A) NASBA Saliva lysatereaction with fluorescence detection 
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							Take 1 μl from the product of Step 1 (saliva lysate) and add into the NASBA reaction mixture (without the enzyme mix) to make a total volume of 15 μl. Reaction mixture can either be prepared in-house or from the Life Sciences NASBA liquid kit (see tables below) using one of the two temperature settings below. 
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							a. Reaction mixture without the enzyme mix is incubated at 65 °C for 2 min followed by a 10-min incubation at 41 °C. Following that, 5 μl enzyme mix is added into the reaction and incubated at 41 °C for a further of 90-120 min. 
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							b. Alternatively, reaction mixture without the enzyme mix is incubated at 95 °C for 5 min followed by a 10-min incubation at 41 °C. Following that, 5 μl enzyme mix is added into the reaction and incubated at 41 °C for a further 90-120 min. 
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							A fluorescence plate reader (e.g. FluoSTAR) can be used to monitor the reaction in real- time, or as an endpoint assay. 
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							  ABCD1 vol.stock conc.conc. in RM2Saliva lysate1 μl  3primers*/beacon# mix1 μl500 nM each primer, 400 nM beacon25 nM each primer, 20 nM beacon4spiked-in viral RNA/water3 μl 5buffer (NECB-24)6.7 μl  6nucleotide (NECN-24)3.3 μl  7enzyme mix (NEC-1-24)5 μl  8total volume20 μl    ABCD1 vol.stock conc.conc. in RM2Saliva lysate1 μl  3primers*/beacon# mix1 μl25 nM each primer 20 nM beacon25 nM each primer 20 nM beacon4spiked-in viral RNA/water4 μl 5buffer with DMSO*5 μl  6nucleotide mix*4 μl  7enzyme mix*5 μl  8total volume20 μl    ABCD1  vol.stock conc.conc. in RM2Tris-HCl pH 8.4*120 μl1 M40 mM3MgCl239.6 μl1 M13.2 mM4KCl112.5 μl2 M75 mM5DTT 30 μl1 M10 mM6DMSO450 μl100%11%7water247.9 μl  8total volume1000 μl    ABCD1  vol.stock conc.conc. in RM2Tris-HCl pH 8.4120 μl1 M40 mM3MgCl239.6 μl1 M13.2 mM4KCl112.5 μl2 M75 mM5DTT 30 μl1 M10 mM6water697.9 μl  7total volume1000 μl    ABCD1  vol.stock conc.conc. in RM2dNTP0.22 μl each100 mM1 mM each3NTP0.88 μl each100 mM4 mM each4total volume4.4 μl    ABCD1  vol.stock conc.conc. in RM2diluted RNase H0.17 μl500 U/ml3.75 U/ml3Photoscript RT 0.28 μl200000 U/ml2500 U/ml4T7 polymerase 2.75 μl50000 U/ml6250 U/ml5BSA0.13 μl20 mg/ml0.12 mg/ml6buffer without DMSO1.78 μl  7water0.40 μl  8total volume5.5 μl    ABC1  vol.stock conc.2RNase H5 μl5000 U/ml3BSA (0.48mg/ml)1.2 μl20 mg/ml4buffer without DMSO16.67 μl 5water27.13 μl 6total volume50 μl (Option B) NASBA reaction with lateral flow dipstick detection For detection with a lateral flow assay, a NASBA lyophilised kit is used with the constitution of the reaction mixture shown below. Take 4 μl from the product of Step 1 (saliva lysate) and add into the NASBA reaction mixture (without the enzyme mix) to make a total volume of 60 μl. Incubate at 95 °C for 5 min followed by a 10-min incubation at 41 °C. Following that, 20 μl enzyme mix is added into the reaction and incubated at 41 °C for a further of 90-120 min. Take the reaction product to the sample well of a PCRD test cassette. Results will be shown within 10 min.
ABCD1 vol.stock conc.conc. in RM2Saliva lysate1 μl  3primers*/beacon# mix1 μl500 nM each primer, 400 nM beacon25 nM each primer, 20 nM beacon4spiked-in viral RNA/water3 μl 5buffer (NECB-24)6.7 μl  6nucleotide (NECN-24)3.3 μl  7enzyme mix (NEC-1-24)5 μl  8total volume20 μl
ABCD1 vol.stock conc.conc. in RM2Saliva lysate1 μl  3primers*/beacon# mix1 μl25 nM each primer 20 nM beacon25 nM each primer 20 nM beacon4spiked-in viral RNA/water4 μl 5buffer with DMSO*5 μl  6nucleotide mix*4 μl  7enzyme mix*5 μl  8total volume20 μl
ABCD1  vol.stock conc.conc. in RM2Tris-HCl pH 8.4*120 μl1 M40 mM3MgCl239.6 μl1 M13.2 mM4KCl112.5 μl2 M75 mM5DTT 30 μl1 M10 mM6DMSO450 μl100%11%7water247.9 μl  8total volume1000 μl
ABCD1  vol.stock conc.conc. in RM2Tris-HCl pH 8.4120 μl1 M40 mM3MgCl239.6 μl1 M13.2 mM4KCl112.5 μl2 M75 mM5DTT 30 μl1 M10 mM6water697.9 μl  7total volume1000 μl
ABCD1  vol.stock conc.conc. in RM2dNTP0.22 μl each100 mM1 mM each3NTP0.88 μl each100 mM4 mM each4total volume4.4 μl
ABCD1  vol.stock conc.conc. in RM2diluted RNase H0.17 μl500 U/ml3.75 U/ml3Photoscript RT 0.28 μl200000 U/ml2500 U/ml4T7 polymerase 2.75 μl50000 U/ml6250 U/ml5BSA0.13 μl20 mg/ml0.12 mg/ml6buffer without DMSO1.78 μl  7water0.40 μl  8total volume5.5 μl
ABC1  vol.stock conc.2RNase H5 μl5000 U/ml3BSA (0.48mg/ml)1.2 μl20 mg/ml4buffer without DMSO16.67 μl 5water27.13 μl 6total volume50 μl
?. Library construction for NGS 
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							To allow for pooled sequencing of NASBA reaction end products, barcode sequences are added upstream of each of the forward and reverse primers (Figure 3a). In addition, an Illumina sequencing adaptor is added upstream of the forward primer barcode sequence as a universal PCR handle (see Materials and reagents section for the exact sequence). Here, 2 μl NASBA end products from each sample are first pooled into a single tube. Pooled products are then column purified to remove residual NASBA primers (QIAquick PCR Purification Kit). PCR is performed on the column purified pooled sample using two NGS indexing primers. Here, we have designed a customised NGS primer containing the T7 polymerase promoter sequence (see Materials and reagents section for the exact sequence) at the P5 end and used a standard TruSeq sequencing primer at the P7 side. A PCR mix is made based on the table below. A standard PCR program is used with longer elongation time and minimal cycle number to reduce barcode hopping.   AB1vol.2															2x PCR mix (KAPA HIFI HotStart ReadyMix)																												20 μl													3															P5 end primer (10 μM)																												1 μl													4															P7 end primer (10 μM)																												1 μl													5															Column purified NASBA product (~3.5 ng dsDNA)																												4 μl													6															Nuclease free water																												14 μl													  ABC1															temperature																												time																												cycle number													2															95 °C																												3 min																												1													3															98 °C																												20 s																												15													4															60 °C																												15 s													5															72 °C																												30 s													6															72 °C																												4 min																												1													After the PCR, an AMPure bead-based double size selection is carried out (0.55x and 0.75x) to enrich for products of interest. In this study, a MiSeq Reagent Kit v2 (300- cycles) was used for NGS.
AB1vol.2															2x PCR mix (KAPA HIFI HotStart ReadyMix)																												20 μl													3															P5 end primer (10 μM)																												1 μl													4															P7 end primer (10 μM)																												1 μl													5															Column purified NASBA product (~3.5 ng dsDNA)																												4 μl													6															Nuclease free water																												14 μl
ABC1															temperature																												time																												cycle number													2															95 °C																												3 min																												1													3															98 °C																												20 s																												15													4															60 °C																												15 s													5															72 °C																												30 s													6															72 °C																												4 min																												1
?. Analysis of NGS results To analyse the INSIGHT NGS data, sequences in FASTQ files are first trimmed to leave the first 80 nucleotides for both read 1 and read 2 using FASTX_trimmer. The trimmed read 1 and paired read 2 are then merged by FLASH. The merged sequence is compared with the reference viral genome sequence (NNNNNACACCTGTGCCTGTTAAACCATTGAAGTTGAAATTGACACATTTGTT TTTAACCAAATTAGTAGACTTTTTAGGTCCACAAACAGTTGCTGGNNNNN, N stands for the barcode position), and only those with a hamming distance less than or equal to 2 are extracted. Here, only substitutions were allowed while insertion- and deletion- containing reads were filtered out. The first 5 nt and the final 5 nt regions of all extracted sequences correspond respectively to the right barcode and the reverse complement of the left barcode. Diagnostic results for sequenced NASBA samples are determined according to the read counts of their corresponding sample-specific barcode pairs. More details can be found in www.github.com/suochenqu/INSIGHT.