Sequencing

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

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
  • 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 400M clusters which translates to ~400M single reads or ~800M paired end reads per run
  • Using P1 Cartridges, 100M clusters are generated on average which translates to ~100M single reads or ~200M paired end reads per run

Read Lengths

The desired read length determines which reagent kit is used:

  • P2 Cartridge with 100 Cycles: up to 50 bp paired end or 100 bp single read and 10X libraries
  • P2 Cartridge with 200 Cycles: up to 100 bp paired end or 150 bp single read
  • P2 Cartridge with 300 Cycles: up to 150 bp paired end
  • P1 Cartridge with 300 Cycles: up to 150 bp paired end or 150 bp single read

Loading Volume and Concentrations

Loading volume is 20 μL

Loading concentration varies by library type:

  • AmpliSeqTM 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
  • 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