The NxSeq™ SARS-CoV-2 Whole Genome Library Kit supplies the targeting primers, enzymes and other reagents necessary to build overlapping, targeted amplicon libraries covering the SARS-CoV-2 genome. These libraries enable whole genome viral sequencing on Illumina instruments for the identification and tracking of SARS-CoV-2 variants. We designed the targeting primer pairs against the Wuhan-Hu-1 strain (NC_045512.2) and carefully screened and optimised the primer set to produce high on-target rates and consistent amplicon yields in order to maximise multiplexing, streamline analysis and keep costs low. Each amplicon target region* overlaps with the target regions of adjacent amplicons in order to generate nearly full coverage and effective variant analysis of each SARS-CoV-2 isolate.
- Identify both known and novel variants: 98% SARS-CoV-2 genome coverage
- Fast, easily automated protocol: 2 hours from cDNA to final library with minimal steps
- Extensive compatibility across Illumina sequencers: Small amplicons enable 2 x 150 PE sequencing chemistry available on more Illumina platforms (compared to amplicon panels >350 bp in size, e.g. ARTIC)
- High multiplexing drives down costs: 1440 CDI indexes plus uniform coverage maximise multiplexing capabilities and simplify analysis.
|Input||Single-stranded or double-stranded cDNA made from positive RNA samples|
|SARS-CoV-2 genome coverage||98% or 29,355 / 29,903 total nucleotides of viral RNA genome|
|Amplicon number||341 overlapping amplicons|
|Amplicon sizes||101 nt average target region* length making it compatible with common 2x150 PE sequencing chemistry|
|Protocol time||2 hours from cDNA to final library|
|Components provided**||SARS-CoV-2 targeting primers, PCR and library prep components|
|Index style||15 Indexing plates are available, each with 96 different combinatorial dual indexes|
|Multiplexing levels||Up to 1440 samples when using all 15 different indexing plates|
|Recommended sequencing depth||~1 million reads per library – may be reduced depending on sample quality|
*Target region refers to the portion of each amplicon between the 3’ ends of each forward and reverse primer pair. This portion of each amplicon will provide the useable variant information, and each target region overlaps with an adjacent target region.
** cDNA synthesis reagents and clean-up/size selection beads are not included.
Simple and robust 2 hour protocol from cDNA to final library
Figure 1. Overview of the NxSeq SARS-CoV-2 Whole Genome Library Kit protocol. This figure illustrates the basic library preparation method used in this targeted amplicon-seq protocol. In total, there are 341 overlapping library amplicons constructed, and in some cases, the forward and reverse primers from adjacent amplicons may produce longer amplicons that span from the 5’ end of the first amplicon to the 3’ end of the downstream adjacent amplicon. Please note that since the final library is not PCR amplified, there will be some fragments in the library that do not have adaptors at both ends and are not sequenceable (not shown).