OneCOVID19NGS: Massively parrallel screening for Covid-19 on NGS platform v1
Authors: L Pedersen Morten 
Published: September 2020

MEASURAND: Specific nucleic acid sequences from the genome of the SARS-CoV-2: SARS-CoV-2 testing utilizes the CDC-developed assay that targets the Nucleocapsid gene of this virus. Two gene targets, N1 and N2, are used to detect cases of COVID-19. During testing of clinical samples, both N1 and N2 nucleocapsid genes must be detected in order for the sample to be determined as a positive. The primer and probe sequences for the N1 and N2 genes, as well as those for the RNase P control sequences, can be found on the CDC homepage: https://www.cdc.gov/coronavirus/2019-ncov/lab/rt-pcr-panel-primer-probes.html. REGULATORY INFORMATION: An EUA filing for this method in under preparation. PROPOSED INTENDED USE: Intended Use: The SARS-CoV-2 assay is a massively parrallel screening for Covid-19 on an NGS platform intended for the qualitative detection of nucleic acid from the SARS-CoV-2 in nasopharyngeal swabs, oropharyngeal swabs, nasal swabs, mid-turbinate nasal swabs, BALs, saliva and sputum from individuals suspected of COVID-19 and for screening a large population for Covid-19. Testing is limited to Genomic Expression that is a Clinical Laboratory Improvement Amendments of 1988 (CLIA), 42 U.S.C. §263a certified high-complexity laboratory. Results are for the detection and identification of SARS-CoV-2 RNA. The SARS-CoV-2 RNA is generally detectable in respiratory specimens during the acute phase of infection. Positive results are indicative of active infection with SARS-CoV-2 but do not rule out bacterial infection or co-infection with other viruses. The agent detected may not be the definite cause of disease. Laboratories within the United States and its territories are required to report all positive results to the appropriate public health authorities. Negative results do not preclude SARS-CoV-2 infection and should not be used as the sole basis for patient management decisions. Negative results must be combined with clinical observations, patient history, and epidemiological information. The assay is intended for use by CLIA certified high-complexity laboratories with experience in developing molecular diagnostics and is only for use under the Food and Drug Administration’s Emergency Use Authorization. Steps: Ordering, shipping and reporting Ordering is done through the ordering portal here: www.covid19.genomicexpression.com Collection kits are shipped to the customer and then sent back to Genomic Expression after collection is carried out. The customer can upload samples in bulk and can follow the progress of the analysis on-line in realtime. After the analysis the customer gets an email notification that the report is ready for download. It takes approximately 5 min to setup a customer profile and order a kit as well as ordeing the analysis of a sample and submitting all PHI for the sample. Returning customers and customers using bulk upload of sample PHI can complete this section in less than 5 min/sample. A bridge study has been initiated in order to qualify VTM products (For the collection kit) from a number of vendors with the ultimate aim of providing a fully integrated and safe home-collection Covid-19 test in the near future. Accessioning The ordering portal is connected to the accessioning portal where the 6-character datamatrix code on the sample vial is scanned to access the patient name and DOB as the customer submitted it and compare that information to the information on the card accompaning the sample. Finally the 6-character datamatrix code on the sample vial is visually verified to be identical to the 6-character datamatrix code on the accompaning card. We're working on automating the accessioning process so that the datamatrix on both the card and the vial are scanned, compared, and verified with minimal human intervention, and where an aliquot of the sample is automatically placed in a labeled (With the same datamatrix code as the intial vial and a running number) microcentrifuge tube or a 96-well plate. No PHI is on the vial as it enters the lab for analysis. All PHI is kept apart from the sample and the only connection is the 6-character datamatrix code that is matched to the analysis by the time of reporting. All accompaning cards are kept for 4 years behind a safe lock. Targeted RNA extraction We're setting up RNA extraction with our own OneRNA kit (Estimated cost of $10/sample from sample to sequencing-ready pool and a pool size where first goal is $1/sample for the sequencing part. I.e. a total of $11 on materials). The extraction uses a capture probe that is compatible with a VTM that inactivates and preserves the SARS-CoV-2 virus by denaturing everything in the sample. The targeted extraction is carried out on the KingFisher platform from ThermoFisher or a simiilar platform with similar capabilities. Analysis The assay is a massively parrallel screening for Covid-19 on an NGS platform intended for the qualitative detection of nucleic acid from the SARS-CoV-2 in a variaty of sample types and intended for screening a large population for Covid-19 within days. The SARS-CoV-2 test kit is designed to detect RNA from the SARS-CoV-2 in respiratory specimens from patients as recommended for testing by public health authority guidelines. The steps involved in abbreviated from: 1.Targeted extraction of SARS-CoV-2 RNA and a human control RNA from sample 2.Preparation of individually labeled libraries, pooling, amplification of the pool with i5 and i7 primers, and sequencing. 3.Collection of data 4.Analysis of data The preparation of individually labeled libraries of the samples constitutes nothing more than an additional step that is integrated into the targeted RNA extraction. Amplification of the pool with i5 and i7 primers and sequencing are standard Illumina procedures. We collect the data on the Illumina BaseSpace platform. For the analysis of the data we're working on an automated analysis pipeline that allows raw sequence to report of the result in minutes. The sequencing run can be cut short because of the size of the target so that the total accessioning-to-report time can be performed witin 1 workday - or even faster. Other targets for e.g. seasonal flu can easily be integrated into the test so that all relevat targets are interrogated all in one go. Assay results and interpretation The test is based upon counting statistics. We're aware of the potential of "index bleeding" to give false positive results at a low rate and are implementing strategies to mitigate this issue. The exact number of reads counted to constitute a positive sample has yet to be determined. But as with a PCR based tests this number should be higher than potential cross contamination counts - or cross contamination should be eliminated. Thus, more testing of the method is needed to answer this question. Inclusivity (analytical sensitivity) Sequences for N1 and N2 primers/probes used in this assay are identical to the N1 and N2 primers/probes sequences used is the FDA authorized CDC SARS-CoV-2 assay.In-silicoanalysis of primers/probes inclusivity was performed by the CDC using all publicly available nucleic acid sequences for 2019-nCoV that were submitted to GenBank as of February 1, 2020. Analysis steps and results are detailed in the CDC 2019-Novel Coronavirus (2019-nCoV) Real-Time RT-PCR Diagnostic Panel Instructions for Use. Cross-reactivity (Analytical Specificity) In-silico BLASTn analysis of primer/probe cross-reactivity was performed by the CDC. Analysis steps and results are detailed in the CDC 2019-Novel Coronavirus (2019-nCoV) Real-Time RT-PCR Diagnostic Panel Instructions for Use.