Sequencing on the Illumina NextSeq 1000
Cartridges:
- The NextSeq 1000 is currently only compatible with Illumina NextSeq 1000/2000 P1 and P2 reagent kits
- Single-use and contains all reagents required for a run including onboard denaturation and dilution
- Library and flow cell load directly into cartridge and are then loaded onto the instrument
Flow Cell:
- Patterned, single-lane glass-based flow cell increases output reads and data
- Each has millions of nanowells where clusters are generated and sequencing reactions are performed
New XLEAP Sequencing by Synthesis Chemistry:
- Higher fidelity polymerase reduces error rates
- Faster base incorporation and block cleavage means faster run times
- Reduced phasing and pre-phasing
- Decreased run costs of up to 20%
Sample Requirements
- All libraries to be sequenced must have Illumina-compatible p5 and p7 sequences that allow the library to bind and generate clusters on the flow cell
- All libraries to be pooled and sequenced in a single run must be multiplexed with unique and compatible indexes
- All libraries and pools submitted must be at least 2 nM in concentration as determined by a fluorometric assay
- Custom read primers and custom index primers can be loaded onto the cartridge
Number of Reads per Run
- Using P2 Cartridges, the NextSeq 1000 can generate an average of 450M clusters which translates to ~450M single reads or ~900M paired end reads per run
- Using P1 Cartridges, 110M clusters are generated on average which translates to ~110M single reads or ~220M paired end reads per run
Sequencing Run Options
| Cartridge & Flow Cell | Clusters Per Run | Number of Cycles | Max. Read Length | Gbp Per Run |
|---|---|---|---|---|
| NextSeq 1000/2000 P1 XLEAP-SBS Reagents | ~100-130 million | 600 | 2 x ~300 bp | > 60 |
| ~100-130 million | 300 | 2 x ~150 bp | > 30 | |
| ~100-130 million | 100 | 2 x ~50 bp | > 10 | |
| NextSeq 1000/2000 P2 XLEAP-SBS Reagents | ~450-530 million | 600 | 2 x ~300 bp | > 180 |
| ~450-530 million | 300 | 2 x ~150 bp | > 120 | |
| ~450-530 million | 200 | 2 x ~100 bp | > 80 | |
| ~450-530 million | 100 | 2 x ~50 bp | > 40 |
Loading Volume and Concentrations
Loading volume is 20 μL
Loading concentration varies by library type:
- AmpliSeq for Illumina Library PLUS - 750 pM
- Illumina DNA Prep - 750 pM
- Illumina DNA Prep with Enrichment - 1000 pM
- Illumina Stranded Total RNA with Ribo-Zero Plus - 750 pM
- Illumina Stranded mRNA Prep - 750 pM
- Illumina DNA PCR-Free - 1000 pM
- TruSeq DNA Nano 350 - 1200 pM
- TruSeq DNA Nano 550 - 1500 pM
- TruSeq Stranded mRNA - 1000 pM
- 10x Genomics 3' or 5' Single-Cell Gene Experession - 650-725 pM
- For other library types, 650 pM is the recommended starting loading concentration
- For libraries/pools of lower starting concentration, dilution and denaturation can be done manually prior to loading