Leveraging the power of long reads for targeted sequencing
December 2024
Authors:
Shruti V. Iyer, Sara Goodwin, and William Richard McCombie
Abstract:
“Long-read sequencing technologies have improved the contiguity and, as a result, the quality of genome assemblies by generating reads long enough to span and resolve complex or repetitive regions of the genome. Several groups have shown the power of long reads in detecting thousands of genomic and epigenomic features that were previously missed by short-read sequencing approaches. While these studies demonstrate how long reads can help resolve repetitive and complex regions of the genome, they also highlight the throughput and coverage requirements needed to accurately resolve variant alleles across large populations using these platforms. At the time of this review, whole-genome long-read sequencing is more expensive than short-read sequencing on the highest throughput short-read instruments; thus, achieving sufficient coverage to detect low-frequency variants (such as somatic variation) in heterogenous samples remains challenging. Targeted sequencing, on the other hand, provides the depth necessary to detect these low-frequency variants in heterogeneous populations. Here, we review currently used and recently developed targeted sequencing strategies that leverage existing long-read technologies to increase the resolution with which we can look at nucleic acids in a variety of biological contexts.”
Sage Science Products:
BluePippin is indicated for the size selection hybridization-based PCR amplicons for PacBio sequencing. The SageHLS with the HLS-CATCH process is described for purifying HMW genomic targets.
Methods Excerpt:
Author Affiliations:
Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.
Genome Research
Resolving the 22q11.2 deletion using CTLR-Seq reveals chromosomal rearrangement mechanisms and individual variance in breakpoints
July 2024
Authors:
Bo Zhou, Carolin Purmann, Hanmin Guo, GiWon Shin, Yiling Huang, Reenal Pattni, Qingxi Meng, Stephanie U. Greer, Tanmoy Roychowdhuryg, Raegan N. Wood, Marcus Ho, Heinrich zu Dohna, Alexej Abyzov, Joachim F. Hallmayer, Wing H. Wong, Hanlee P. Ji, and Alexander E. Urban
Abstract:
“We developed a generally applicable method, CRISPR/Cas9-targeted long-read sequencing (CTLR-Seq), to resolve, haplotype-specifically, the large and complex regions in the human genome that had been previously impenetrable to sequencing analysis, such as large segmental duplications (SegDups) and their associated genome rearrangements. CTLR-Seq combines in vitro Cas9-mediated cutting of the genome and pulse-field gel electrophoresis to isolate intact large (i.e., up to 2,000 kb) genomic regions that encompass previously unresolvable genomic sequences. These targets are then sequenced (amplification-free) at high on-target coverage using long-read sequencing, allowing for their complete sequence assembly. We applied CTLR-Seq to the SegDup-mediated rearrangements that constitute the boundaries of, and give rise to, the 22q11.2 Deletion Syndrome (22q11DS), the most common human microdeletion disorder. We then performed de novo assembly to resolve, at base-pair resolution, the full sequence rearrangements and exact chromosomal breakpoints of 22q11.2DS (including all common subtypes). Across multiple patients, we found a high degree of variability for both the rearranged SegDup sequences and the exact chromosomal breakpoint locations, which coincide with various transposons within the 22q11.2 SegDups, suggesting that 22q11DS can be driven by transposon-mediated genome recombination. Guided by CTLR-Seq results from two 22q11DS patients, we performed three-dimensional chromosomal folding analysis for the 22q11.2 SegDups from patient-derived neurons and astrocytes and found chromosome interactions anchored within the SegDups to be both cell type-specific and patient-specific. Lastly, we demonstrated that CTLR-Seq enables cell-type specific analysis of DNA methylation patterns within the deletion haplotype of 22q11DS.”
Sage Science Products:
SageHLS with the HLS-CATCH process for purifying HMW genomic targets.
Author Affiliations:
Department of Psychiatry and Behavioral Sciences, Stanford University School of Medicine, Stanford, CA Stanford Maternal and Child Health Research Institute, Stanford University School of Medicine, Stanford, CA
Department of Genetics, Stanford University School of Medicine, Stanford, CA
Department of Statistics, Stanford University, Stanford, CA
Department of Biomedical Data Science, Stanford University School of Medicine, Stanford, CA
Division of Oncology, Department of Medicine, Stanford University School of Medicine, Stanford, CA
Division of Computational Biology, Department of Quantitative Health Sciences, Mayo Clinic, Rochester, MN
Department of Biology, American University of Beirut, Lebanon
Program on Genetics of Brain Function, Stanford Center for Genomics and Personalized Medicine, Stanford University School of Medicine, Stanford, CA
CTLR-Seq Protocol
July 2024
Authors:
Bo Zhou, GiWon Shin,Yiling Huang, Raegan N. Wood, Hanlee P. Ji, Alexander E. Urban
Abstract:
“We developed a generally applicable method CRISPR/Cas9-targeted long read sequencing (CTLR-Seq) to resolve, haplotype-specifically, and at base-pair resolution, large, complex, and highly repetitive genomic regions that had been previously impenetrable to next-generation sequencing analysis, i.e. large segmental duplication (SegDup) regions and their associated genome rearrangements that often stretch hundreds of kilobases. CTLR-Seq combines in vitro Cas9-mediated cutting of the genome and pulse-field gel electrophoresis to haplotype-specifically isolate intact large (100-2000 kb) regions that encompass previously unresolvable genomic sequences. These targets are then sequenced (amplification-free) with up to 250x on-target coverage using nanopore sequencing, allowing for their complete sequence assembly.”
Sage Science Products:
SageHLS with the HLS-CATCH process for purifying HMW genomic targets.
Author Affiliations:
Department of Psychiatry and Behavioral Sciences and Department of Genetics, Stanford University School of Medicine, Stanford, CA
Stanford Child Health Research Institute, Stanford University School of Medicine, Stanford, CA
Division of Oncology, Department of Medicine, Stanford University School of Medicine, Stanford, CA
Protocols.io
DOI: 10.17504/protocols.io.q26g71d7kgwz/v1
Processing frozen archival human DNA samples for large-scale SQK-LSK114 Oxford Nanopore long-read DNA sequencing SOP v1
June 2024
Authors:
Alicia Wenghöfer, Kimberly Paquette, Laksh Malik, Breeana Baker, Cedric Kouam, Kimberley J Billingsley
Abstract:
“As part of the GP2 monogenic network we will generate long-read sequencing data to better understand the genetic architecture of Parkinson’s disease. To generate this large-scale Nanopore data we have developed a protocol for processing and long-read sequencing frozen human DNA samples, ideally targeting an N50 of ~30kb and ~30X coverage. However, with archival human DNA samples, we usually see a drop in DNA quality in terms of DNA length. Therefore, this protocol is focused on attempting to achieve the highest N50 and coverage possible from the input available.“
Sage Science Products:
BluePippin with High-Pass Plus gel cassettes, >10kb High-Pass protocol.
Author Affiliations:
Center for Alzheimer’s and Related Dementias, National Institute on Aging, Bethesda, Maryland
Institute of Neurogenetics, University Hospital Schleswig-Holstein, Luebeck, Germany
Laboratory of Neurogenetics, National Institute on Aging, Bethesda, Maryland
Protocols.io
DOI: 10.17504/protocols.io.5jyl82morl2w/v1
Identifying the best PCR enzyme for library amplification in NGS
April 2024
Authors:
Michael A. Quail, Craig Corton, James Uphill, Jacqueline Keane and Yong Gu
Abstract:
“PCR amplification is a necessary step in many next-generation sequencing (NGS) library preparation methods. Whilst many PCR enzymes are developed to amplify single targets efficiently, accurately and with specificity, few are developed to meet the challenges imposed by NGS PCR, namely unbiased amplification of a wide range of different sizes and GC content. As a result PCR amplification during NGS library prep often results in bias toward GC neutral and smaller fragments. As NGS has matured, optimized NGS library prep kits and polymerase formulations have emerged and in this study we have tested a wide selection of available enzymes for both short-read Illumina library preparation and long fragment amplification ahead of long-read sequencing.
We tested over 20 different hi-fidelity PCR enzymes/NGS amplification mixes on a range of Illumina library templates of varying GC content and composition, and find that both yield and genome coverage uniformity characteristics of the commercially available enzymes varied dramatically. Three enzymes Quantabio RepliQa Hifi Toughmix, Watchmaker Library Amplification Hot Start Master Mix (2X) ‘Equinox’ and Takara Ex Premier were found to give a consistent performance, over all genomes, that mirrored closely that observed for PCR-free datasets. We also test a range of enzymes for long-read sequencing by amplifying size fractionated S. cerevisiae DNA of average size 21.6 and 13.4 kb, respectively.
The enzymes of choice for short-read (Illumina) library fragment amplification are Quantabio RepliQa Hifi Toughmix, Watchmaker Library Amplification Hot Start Master Mix (2X) ‘Equinox’ and Takara Ex Premier, with RepliQa also being the best performing enzyme from the enzymes tested for long fragment amplification prior to long-read sequencing. “
Sage Science Products:
BluePippin and SageELF were used to fractionate DNA for testing long-range PCR enzymes.
Methods Excerpt:
“Sheared S. cerevisiae DNA was size fractionated using Sage Sciences ELF or Bluepippin instruments yielding modal fragment sizes of 21.6 and 13.3 kb, respectively. After adapter ligation 1 ng of each of these were used as a template for long range PCR with a range of enzymes using manufacturers recommended cycling conditions…”
Author Affiliations:
Wellcome Sanger Institute, Hinxton, Cambs.,UK
Department of Medicine, University of Cambridge, Cambridge, Cambs., UK
Microbial Genomics