Instrumentation

Illumina NovaSeq X Plus

The Illumina NovaSeq X Plus was released in the spring of 2023 and represents the next evolution of their flagship Sequencing by Synthesis (SBS) technology with the incorporation of XLEAP-SBS chemistry. Similar to many instruments from Illumina this system is highly flexible with two different sequencing positions that can each independently sequence one of the compatible flow cell types (1.5B, 10B, 25B). The different flow cell types with 100, 200, or 300 chemistry cycles allows for the selection of the most optimal sequencing configuration for your application and needed turnaround time. To optimize these factors at TGen we recommend most applications target the use of 10B 300 cycle lanes for the best turnaround time and cost. For the most cost effective approach for most applications we recommend 25B 300 cycle lanes. For counting applications that do not require long read lengths like RRBS or certain single cell applications we recommend 10B 100 cycle flow cells, which have 138 total cycles available but you must submit pools to fill all 8 lanes. If you can not fill an entire flow cell, the recommendation would be 10B or 25B 300 cycle lanes.

Important Features:

• Industry leading cost per read and cost per Gb
• Fast sequencing, <24 hours for 1.5B and 10B flow cells and 48 hours for 25B flow cells
• Minimizes output file size by binning quality scores (Q12, Q24, and Q40)
• Improved based accuracy with >85% of output bases binned at Q40 with the majority of these bases exceeding Q40 without binning. 

PacBio Onso

The PacBio Onso was released in the summer of 2023 and TGen received the first production instrument in August 2023. This short read sequencer uses Sequencing by Binding (SBB) technology to achieve industry leading base call accuracy with >85% of bases exceeding Q40 and median base qualities exceeding Q50, one error in 100,000 bases. The flow cells have two independently addressed lanes that together can produce 800-1000 million paired-end reads using 100x100 or 150x150 sequencing formats. There is also a single read 200 cycle option available. Onso compatible libraries can be generated directly using Onso compatible adaptors, alternatively Illumina compatible libraries can be converted into Onso compatible libraries.

Important Features:

• Industry leading base call accuracy
• Accurate reads through homopolymers
• Low loading concentration facilitates lower inputs 

PacBio Revio

The PacBio Revio was released in the spring of 2023 and provides high volume accurate long read sequencing using circular consensus sequencing (CCS) to produce HiFi long reads. The instrument has 4 independent sequencing positions for individual SMRT-cells that each have 25 million zero-mode waveguide (ZMW) sequencing wells. A single SMRT-cell is capable of producing >30x sequence coverage of a human genome after 24 hours of sequencing. Beyond producing long, high-accuracy sequencing reads when libraries are constructed from unmanipulated DNA you also get 5mC methylation of CpG sites in eukaryotes or 4mC and 6mA methylation in prokaryotes. Finally, using KINNEX array kits you can cost effectively perform full length RNA isoform analysis from bulk RNA or single cell RNA and complete 16S ribosomal RNA analysis of microbial populations.

Important Features:

• Accurate HiFi reads created using circular consensus sequencing and Google DeepConsensus providing gold standard long-read base qualities for genome assembly and other applications
• Improved platform eliminates the need for problematic processing steps
• Option for different movie times (16, 24, 30), with 30 hour movie supporting HiFi read generation for molecules in the 20-25 kb range.

Oxford Nanopore PromethION-24

The Oxford Nanopore PromethION-24 with an A100 compute tower was released in the fall of 2022 and provides high-throughput nanopore sequencing with median based qualities >Q20 on simplex reads and >Q30 on duplex reads. The instrument has 24 independent sequencing positions for promethION flow cells that each have 2675 sequencing channels with 4 independent sequencing pores that are active at any one point in time. This instrument is highly flexible with the ability to sequencing native DNA fragments in standard ligation assays with N50 molecule lengths between 30-60kb and exceeding 100kb with ultra-long protocols. For maximum data production DNA can be fragmented to target ranges of 10-30kb, which typically results in human genomes with 40-60x coverage. Additionally, since native DNA is analyzed, modified 5-methyl cytosine and 5-hydroxymethyl cyctosine bases are identified providing epigenetic measurements in human samples while for prokaryotes 6mA can also be detected. Sequencing effort can be focused on specific regions of the genome using adaptive sampling where the first 500-1000 bp of a read is interrogated for target matching to determine if a molecule will be sequenced further.

Important Features:

• Highly versatile platform with 24 independent sequencing positions facilitating the sequencing of >2900 human genomes a year
• Supports multiple applications from ultra-long DNA analysis to direct RNA sequencing
• Option for adaptive sampling to improve coverage of regions of interest that can be enabled across the flow cell or only in specific quadrants.
• Single read accuracy exceeds Q20 and improves with advances in base calling without a need to resequence
• Duplex read accuracy exceeds Q30, with standard runs producing 20% duplex reads and high duplex flowcells producing >80% duplex reads
• Both 5mC and 5hmC base modifications can be detected in human samples