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SRX10605608: GSM5242307: SARS-CoV-2 titration - 10000 - rep2; Homo sapiens; OTHER
1 ILLUMINA (Illumina NovaSeq 6000) run: 301,675 spots, 30.2M bases, 6.9Mb downloads

Submitted by: NCBI (GEO)
Study: COV-ID: A LAMP sequencing approach for high-throughput co-detection of SARS-CoV-2 and influenza virus in human saliva
show Abstracthide Abstract
We combined RT-LAMP with deep sequencing to detect as few as 5–10 virions of SARS-CoV-2 in unprocessed human saliva. Based on a multi-dimensional barcoding strategy, COV-ID can be used to test thousands of samples overnight in a single sequencing run with limited labor and laboratory equipment. The sequencing-based readout allows COV-ID to detect multiple amplicons simultaneously, including key controls such as host transcripts and artificial spike-ins, as well as multiple pathogens. Here we demonstrate this flexibility by simultaneous detection of 4 amplicons in contrived saliva samples: SARS-CoV-2, influenza A, human STATHERIN, and an artificial SARS spike-in. The approach was validated on clinical saliva samples, where it showed 100% agreement with RT-qPCR. COV-ID can also be performed directly on saliva adsorbed on filter paper, simplifying collection logistics and sample handling. Overall design: Water or human saliva was spiked with synthetic SARS-CoV-2 RNA or influenza A RNA at various concentrations. Modified set of RT-LAMP primers including sample-specific barcodes as well as landing sites for universal Illumina adapters were used to perform various RT-LAMP reactions in multiplex. Different barcoded and amplified samples were pooled and Illumina adapter sequences introduced by PCR. The resulting pools were sequenced and analyzed.
Sample: SARS-CoV-2 titration - 10000 - rep2
SAMN18746794 • SRS8706902 • All experiments • All runs
Organism: Homo sapiens
Library:
Instrument: Illumina NovaSeq 6000
Strategy: OTHER
Source: TRANSCRIPTOMIC
Selection: other
Layout: SINGLE
Construction protocol: We prepared 100x TCEP/EDTA buffer (250 mM TCEP, 100 mM EDTA, 1.15 N NaOH) 29. TCEP/EDTA buffer was added to human saliva at 1:100 volume, then samples were capped, vortexed to mix and heated in a thermocycler (95ºC 5 min, 4ºC hold) until ready to use for RT-LAMP. 1 μL of diluted LAMP material was used as a template for PCR using OneTaq DNA polymerase (NEB Cat. M0480L) with 100 nM each of custom dual-indexed Illumina P5 and P7 primers in either 10 or 25 μL reaction. PCR reactions were incubated as follows: (25 cycles of stage 1 [94ºC x 15 sec, 45ºC x 15 sec, 68ºC x 10 sec], 10 cycles of Stage 2 [ 94ºC x 15 sec, 68ºC x 10 sec], 68ºC x 1 min, 4ºC x ∞).
Experiment attributes:
GEO Accession: GSM5242307
Links:
Runs: 1 run, 301,675 spots, 30.2M bases, 6.9Mb
Run# of Spots# of BasesSizePublished
SRR14242955301,67530.2M6.9Mb2021-04-16

ID:
14082612

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