Efficient deep sequencing and rapid genomic speciation of RNA viruses (vRNAseq)

Patent metadata

Field	Value
Publication number	US-9909176-B2
Application number	US-201514841981-A
Country	US
Kind code	B2
Filing date	Sep 1, 2015
Priority date	Sep 8, 2014
Publication date	Mar 6, 2018
Grant date	Mar 6, 2018

Abstract

A method for limited input whole genome sequencing of RNA viruses includes isolating a viral RNA sample, converting the viral RNA sample to a double-stranded viral cDNA sample, constructing a double-stranded viral cDNA amplicon library from the double-stranded viral cDNA sample, and sequencing the double-stranded viral cDNA amplicon library to obtain a double-stranded viral cDNA sample sequencing read.

First claim

That which is claimed:

1. A method for limited input whole genome sequencing of orthomyxoviruses, noroviruses, flaviviruses, or ebola viruses, the method comprising:
isolating a orthomyxovirus, norovirus, flavivirus, or ebola virus viral RNA sample;
converting the viral RNA sample to a double-stranded viral cDNA sample, wherein the converting includes:
priming first strand cDNA synthesis using an oligonucleotide primer specific for a highly conserved region of a viral genome, wherein the highly conserved region is common to all subtypes of the viral genome as a means of replication,
synthesizing a second cDNA strand with RNase H, DNA ligase, and DNA polymerase I to form the double-stranded viral cDNA sample, and
purifying the double-stranded viral cDNA sample;

constructing a double-stranded viral cDNA amplicon library from the double-stranded viral cDNA sample; and
sequencing the double-stranded viral cDNA amplicon library to obtain a double-stranded viral cDNA sample sequencing read.

2. The method according to claim 1 , wherein isolating the viral RNA sample comprises:
extracting RNA from a supernatant to form an RNA extract sample; and
depleting DNA from the RNA extract sample.

3. The method according to claim 2 , wherein extracting RNA from the supernatant comprises magnetic bead-based nucleic acid isolation.

4. The method according to claim 2 , wherein the supernatant comprises about 10 3 RNA viruses.

5. The method according to claim 2 , wherein the supernatant comprises an in vivo supernatant.

6. The method according to claim 1 , wherein constructing the double-stranded viral cDNA amplicon library comprises:
performing tagmentation reactions on the double-stranded viral cDNA sample to obtain the double-stranded viral cDNA amplicon library;
purifying the double-stranded viral cDNA amplicon library;
quantifying the double-stranded viral cDNA amplicon library; and
pooling multiplexed double-stranded viral cDNA amplicon libraries.

7. The method according to claim 6 , wherein one set of primers is effective in performing tagmentation reactions across different strains within a viral family.

8. The method according to claim 1 , wherein sequencing the double-stranded viral cDNA amplicon library comprises:
denaturing the double-stranded viral cDNA amplicon library;
loading the double-stranded viral cDNA amplicon library onto a sequencer; and
running the sequencer to obtain the double-stranded viral cDNA sample sequencing read.

9. The method according to claim 1 , wherein sequencing the double-stranded viral cDNA amplicon library comprises sequencing from about 0.1 pg to about 10 pg converted viral genomes.

10. The method according to claim 1 , further comprising analyzing the double-stranded viral cDNA sample sequencing read with an ultrafast read classifier.

11. The method according to claim 10 , wherein analyzing the double-stranded viral cDNA sample sequencing read with an ultrafast read classifier further comprises:
querying at least one database; and
identifying the double-stranded viral cDNA sample sequencing read as belonging to an RNA viral species.

12. The method according to claim 11 , wherein the at least one database comprises at least one of a full metagenomics implementation, a pan-virus implementation, a virus-specific implementation, or any combination thereof.

13. The method according to claim 10 , wherein the ultrafast read classifier analyzes from about 3 million reads to about 4 million double-stranded viral cDNA sample sequencing reads in from about 1 minute to about 5 minutes and the ultrafast read classifier has a sequence alignment with existing reference genomes of from about 90% to about 100%.

14. The method according to claim 10 , wherein the ultrafast read classifier identifies nearest neighbor RNA viruses in from about 1 minute to about 15 minutes.

15. The method according to claim 1 , wherein the RNA viruses comprise non-segmented genomes.

16. The method according to claim 1 , wherein the RNA viruses comprise negative-sense single-stranded RNA viruses.

17. The method according to claim 1 , wherein the method comprises a completion time of from about 5 hours to about 15 hours and a hands-on time of from about 0.5 hours to about 5 hours.

18. The method according to claim 1 , wherein the supernatant comprises an in vitro supernatant.

19. The method according to claim 1 , wherein the RNA viruses comprise segmented genomes.

20. The method according to claim 1 , wherein the RNA viruses comprise double-stranded RNA viruses.

Assignees

• Univ Johns Hopkins

Inventors

• Thielen Peter M
• Evans Jared D
• Mehoke Thomas S
• Wolfe Joshua T

Classifications

• C12Q1/6874 Primary

  involving nucleic acid arrays, e.g. sequencing by hybridisation · CPC title

• C12Q1/70

  involving virus or bacteriophage {(immunoassay for viruses G01N33/56983)} · CPC title