Library preparation options for sequencing the coronavirus SARS CoV 2 (associated with COVID 19)
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
- 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
- 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
- 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:
- Viral RNA is extracted with the QIAGEN QIAmp Viral Mini Kit (Catalog No. 52904).
- Refer to the AmpliSeq for Illumina Community Panel Reference Guide, Chapter 3, for the Protocol for RNA Panels.
- 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
This panel is intended for research use only (RUO) applications, and not for use in diagnostic procedures.
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