High Throughput Ultra Long Sequencing? The Longest Sequencing Data Ever Generated? Try to Beat Our Record!
High throughput and ultra long reads (100 kb - 1+ Mb) now go hand in hand and can be frequent guests in your lab with Nanobind Ultra Long Sequencing. This method has been used to generate the longest sequencing data in the world. We broke the previous 2.3 Mb record in Jan 2020 with a 2.44 Mb read. A few months later, we broke that record with a 2.9 Mb read. Most recently, Oxford Nanopore broke our record with a 4.1 Mb read of their own.
The Highest Read Length and Throughput Combinations Anywhere!
Compared to previous ultra long sequencing approaches, this new method generates 10-100X greater throughput. Consistently obtain longest reads of 1 - 2+ Mb, read length N50 of 50 - 100+ kb, and throughputs of 10 - 20+ Gb on MinION and 50 - 100+ Gb on PromethION across a wide array of sample types.
This method is designed to maximize the amount of data in 100+ kb and 200+ kb reads. While the method can be used to generate read length N50 in excess of 200 kb, ultra long data throughput is actually maximized with more moderate read length N50 of 50 - 100 kb. The secret is maintaining high pore occupancy.
First, the appropriate Nanobind Big DNA Kit (CBB, Plant Nuclei, or Tissue) is used to extract UHMW (50 kb - 1+ Mb) DNA. We've developed brand new UHMW DNA extraction protocols that result in less viscous DNA, higher sequencing throughputs, and even longer read lengths. Each Nanobind UL Library Prep Kit comes with an UHMW DNA Aux Kit that is used alongside the appropriate Nanobind Big DNA Kit for UHMW DNA extraction.
Then, the Nanobind UL Library Prep Kit is used in conjunction with the Oxford Nanopore Ultra-Long DNA Sequencing Kit (SQK-ULK001) to prepare the UHMW DNA for sequencing. A transposase-based approach is used to add sequencing adapters and motor proteins to the UHMW DNA. The ONT kit contains all the enzymes and reagents. Our kit contains Nanobind disks and buffers to perform reaction cleanup. Unlike magnetic particles, our magnetic Nanobind disks enable rapid purification of megabase DNA without shearing, first in the extractions and then in library prep.
In contrast to other ultra long sequencing approaches, this new approach does not use the Rapid Sequencing Kit (SQK-RAD004). A ground up optimization of extraction, library prep, and sequencing was performed in collaboration with Oxford Nanopore to achieve the high pore occupancies that underlie that massive throughput increases. It is fully compatible with MinION, GridION, and PromethION and responds well to nuclease flush.
The standard Nanobind Ultra Long Sequencing Protocol generates a 6X MinION library or a 3x PromethION library that is split and sequentially loaded onto the same flow cell, interspaced by nuclease flush to maximize throughput (3 loads per flow cells, 24h each). The same protocol is used for MinION/GridION/PromethION.
This protocol was initially developed on cultured cell, bacteria, human blood, and nucleated blood samples. Since then it has been extended to animal tissue and plant samples. Work is ongoing to improve read lengths and further expand sample type.
Each kit can be used to run 6 flow cells using 3X libraries.
Also includes 1 UHMW DNA Aux Kit that is used in conjunction with the appropriate Nanobind Big DNA Kit to perform UHMW DNA extraction. Each Aux Kit contains: 1) 10X RBC Lysis Buffer, 2) Proteinase K, 3) RNase A, 4) CLE3 Digestion Buffer, 5) ULL Lysis/Binding Buffer, and 6) CS Digestion Supplement.
Oxford Nanopore's version of the ultra long library preparation protocol is available here. The processing steps are identical to ours but it contains more guidance on loading/flushing and little less guidance on sample prep.