Abstract:
Structural variants are increasingly recognized as drivers of genetic disease. Recent studies using long-read sequencing have found SVs to be 5-10X more prevalent than previously thought. Whereas germline SVs are readily detected using blood or saliva, somatic SVs can only be detected using tissues. Tissues contain extracellular material that must be efficiently disrupted without damaging delicate HMW DNA.
We present a high throughput workflow for long-read sequencing of clinical tissue specimens. First, Covaris cryoPREP automated dry pulverizer is used to cryofracture frozen tissue samples in tissueTUBEs. Compared to manual methods such LN2 grinding, cryoPREP can cryofracture large numbers of samples with high reproducibility, no cross-contamination, and low user intervention. Then, Circulomics Nanobind magnetic disks are used to extract HMW DNA from the cryofractured tissue powder. Each Nanobind disk is covered with nanostructured silica that protects DNA from shearing to enable higher DNA size, extraction yield, and purity than competing methods. Finally, Covaris AFA technology and Circulomics Short Read Eliminator can be used to shape read length distributions by controllably shearing HMW DNA and
depleting unwanted short fragments. This rapid and reproducible method is easily scaled to accommodate large scale studies of SVs and to underpin future clinical tests.
This method was tested on a comprehensive panel of clinically relevant human tissues including breast, kidney, colon, uterine fibroid, pancreas, and skeletal muscle. DNA size up to 300 kb was obtained across all tissue types with size surpassing 500 kb for some samples. Extraction yields varied across tissue type but were consistent across replicates (11% CV). The DNA were then sequenced on Oxford Nanopore GridION. High read length N50 over 36 kb, throughputs up to 9 Gb, and longest reads surpassing 230 kb were obtained. These high quality DNA samples are suitable for diverse platforms including PacBio, 10X Genomics, and Bionano Genomics.
Structural variants are increasingly recognized as drivers of genetic disease. Recent studies using long-read sequencing have found SVs to be 5-10X more prevalent than previously thought. Whereas germline SVs are readily detected using blood or saliva, somatic SVs can only be detected using tissues. Tissues contain extracellular material that must be efficiently disrupted without damaging delicate HMW DNA.
We present a high throughput workflow for long-read sequencing of clinical tissue specimens. First, Covaris cryoPREP automated dry pulverizer is used to cryofracture frozen tissue samples in tissueTUBEs. Compared to manual methods such LN2 grinding, cryoPREP can cryofracture large numbers of samples with high reproducibility, no cross-contamination, and low user intervention. Then, Circulomics Nanobind magnetic disks are used to extract HMW DNA from the cryofractured tissue powder. Each Nanobind disk is covered with nanostructured silica that protects DNA from shearing to enable higher DNA size, extraction yield, and purity than competing methods. Finally, Covaris AFA technology and Circulomics Short Read Eliminator can be used to shape read length distributions by controllably shearing HMW DNA and
depleting unwanted short fragments. This rapid and reproducible method is easily scaled to accommodate large scale studies of SVs and to underpin future clinical tests.
This method was tested on a comprehensive panel of clinically relevant human tissues including breast, kidney, colon, uterine fibroid, pancreas, and skeletal muscle. DNA size up to 300 kb was obtained across all tissue types with size surpassing 500 kb for some samples. Extraction yields varied across tissue type but were consistent across replicates (11% CV). The DNA were then sequenced on Oxford Nanopore GridION. High read length N50 over 36 kb, throughputs up to 9 Gb, and longest reads surpassing 230 kb were obtained. These high quality DNA samples are suitable for diverse platforms including PacBio, 10X Genomics, and Bionano Genomics.
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