Platforms

NextGen Sequencing Technologies

NISC currently employs the following NextGen sequencing technologies:

Illumina NovaSeq 6000
Illumina NextSeq 550
Illumina MiSeq
PacBio Sequel II
Oxford Nanopore GridION and PromethION

Illumina NovaSeq 6000

The NovaSeq 6000 offers scalable sequencing through multiple flow cell modules. The most cost-effective module is the S4 which produces ~2.5 Tb per flow cell or ~ 625 Gb per lane. Due to the extremely high quantity of sequence output, this technology is primarily useful for projects requiring a very large number of reads, such as whole genome, whole exome and multiplexed transcriptome experiments.

Illumina NextSeq 550

The NextSeq 550 is a mid-range output sequencer. Multiple run options are offered, with outputs ranging from 16 Gb to 120 Gb and read lengths from 1 x 75 b to 2 x 150 b. This platform is ideal for smaller projects or those requiring non-standard run conditions, such as single-cell RNA-Seq.

Illumina MiSeq

MiSeq is a small-scale benchtop sequencer. This instrument is compatible with any of the Illumina libraries described above. MiSeq produces reads up to 300 bases in length from a paired-end read run (2x300 b) with a yield of ~10 Gb. This capability is primarily useful for sequencing microbial genomes and amplicons.

PacBio Sequel II

The PacBio Sequel II platform generates long read lengths while maintaining high consensus accuracy and unbiased coverage. The maximum read length is more than 50 kb and high fidelity (HiFi) reads >10 kb with >99.9% accuracy can also be generated. This platform can generate sequence reads that support high-quality assemblies from small bacterial genomes all the way up to diploid human genomes. Other applications include amplicons, full length 16s sequencing, epigenomes, and RNA isoforms. For most applications one SMRT cell generates 20-50 Gb of data and completes in 20-30 hours.

Oxford Nanopore GridION and PromethION

The Oxford Nanopore platform is the newest and most rapidly evolving platform in our portfolio of sequencing technologies. Monitoring current fluctuations as native DNA or RNA molecules pass through nanopores, this approach has key advantages of being able to read ultra-long fragments (some > 1Mb) and detecting modified bases without special library construction. Base calling is done in real time making sequence data available right from the start of the run. Raw read accuracy is currently about 98% with new advances expected to push that over 99%. The primary uses of this technology to date at NISC have taken advantage of the ultra-long reads to detect large structural variants and create de novo genome assemblies. Using this approach, a typical PromethION flowcells yields 80 Gbase of quality reads with N50 approximately 72kb. Preliminary experiments have also been undertaken to explore direct RNA and full-length cDNA sequencing.