Illumina offers three approaches to help researchers with sequencing
of SARS-CoV-2.
Next-generation sequencing (NGS) provides an effective way to screen
samples and characterize viruses without prior knowledge of the
infectious agent. The Illumina SARS-CoV-2 workflows described below
are not intended for diagnostic purposes (this bulletin does not
address the COVIDSeq workflow). Application notes that provide
detailed information and example results are included for each
workflow, below.
Total RNA sequencing with the TruSeq™ Stranded Total RNA Gold
library preparation workflow
-
Comprehensive
workflow for detecting coronavirus using Illumina benchtop
systems application note.
- Sample preparation workflow:
- Viral RNA is extracted with the QIAGEN QIAmp Viral Mini
Kit (Catalog No. 52904).
- Libraries are prepared
with TruSeq
Stranded Total RNA Library Prep Gold kit (Illumina,
48-sample kit, catalog no. 20020598; 96-sample kit, Catalog No.
20020599).
- Sequencing and data analysis:
- Samples prepared directly from swabs or similar sample
sources are most suited for the NextSeq™ series of systems due
to the recommended 10 million reads per sample.
- Libraries prepared from viral culture using the same library
preparation workflow are well suited for other benchtop
instruments including the iSeq™ 100, MiniSeq™, and MiSeq™
systems due to the lower recommended read count of 500,000 reads
per sample.
- Local data analysis is performed with the
Illumina Local Run Manager (LRM) Resequencing Module, with the
SARS-CoV-2 reference genome.
- Downstream analyses can
be performed with the IDbyDNA Explify platform (www.idbydna.com/explify-platform).
Enrichment-based sequencing with the Illumina DNA Prep with
Enrichment (formerly known as Nextera™ Flex for Enrichment) library
preparation workflow
-
Enrichment
workflow for detecting coronavirus using Illumina NGS systems
application note
- Sample preparation workflow:
- Viral RNA is extracted with the QIAGEN QIAmp Viral Mini Kit
(Catalog No. 52904).
- Viral RNA is reverse
transcribed using Thermo Fisher's Scientific Maxima H Minus
Double-Stranded cDNA Synthesis Kit (Thermo Scientific, Catalog
No. K2561). Other reverse transcription workflows may be
compatible, including the first strand synthesis module and
second strand (nonstranded) synthesis module from NEB.
- Viral cDNA is used as input for the Illumina
DNA Prep with Enrichment library preparation kit (formerly
known as Nextera Flex for Enrichment), and enriched with the Respiratory
Virus Oligo Panel (Illumina, Catalog No. 20042472).
- Sequencing and data analysis:
- Sequencing
is performed on the benchtop iSeq 100, MiniSeq, or MiSeq systems
that are well suited for the low read requirements for these
samples
- Local data analysis is performed with the
Illumina Local Run Manager (LRM) Resequencing Module, with the
SARS-CoV-2 reference genome.
- Downstream analyses can
be performed with the IDbyDNA Explify platform (www.idbydna.com/explify-platform).
Amplicon sequencing with the AmpliSeq™ for Illumina SARS-CoV-2
Research Panel
-
AmpliSeq
for Illumina SARS-CoV-2 Research Panel product page
- Panel overview:
- This AmpliSeq community
panel (made-to-order) contains 247 amplicons in two pools
targeting the SARS-CoV-2 genome. The panel is designed for
>99% coverage of the SARS-CoV-2 genome (~30 kb) and covers
all potential serotypes.
- In addition to the community
panel, an AmpliSeq for Illumina Library
PLUS kit, Indexes, and cDNA Synthesis kit are required to
generate libraries.
- Sample preparation
workflow:
- Sequencing and data analysis:
- After library
preparation, sequencing is typically performed on
the benchtop sequencers, iSeq 100, MiSeq, MiniSeq, or NextSeq
500/550 due to the low read requirements for these samples
- The DNA
Amplicon app in BaseSpace™ or the DRAGEN™
Metagenomics app can be used for data analysis.
This panel is intended for research use only (RUO) applications,
and not for use in diagnostic procedures.