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
Whole genome sequencing identifies associations for nonsyndromic sagittal craniosynostosis with the intergenic region of BMP2 and noncoding RNA gene LINC01428
April 2024
Authors:
Anthony M. Musolf, Cristina M. Justice, Zeynep Erdogan-Yildirim, Seppe Goovaerts, Araceli Cuellar, John R. Shaffer, Mary L. Marazita, Peter Claes, Seth M. Weinberg, Jae Li, Craig Senders, Marike Zwienenberg, Emil Simeonov, Radka Kaneva, Tony Roscioli, Lorena Di Pietro, Marta Barba, Wanda Lattanzi, Michael L. Cunningham, Paul A. Romitti & Simeon A. Boyadjiev
Abstract:
“Craniosynostosis (CS) is a major birth defect resulting from premature fusion of cranial sutures. Nonsyndromic CS occurs more frequently than syndromic CS, with sagittal nonsyndromic craniosynostosis (sNCS) presenting as the most common CS phenotype. Previous genome-wide association and targeted sequencing analyses of sNCS have identified multiple associated loci, with the strongest association on chromosome 20. Herein, we report the first whole-genome sequencing study of sNCS using 63 proband-parent trios. Sequencing data for these trios were analyzed using the transmission disequilibrium test (TDT) and rare variant TDT (rvTDT) to identify high-risk rare gene variants. Sequencing data were also examined for copy number variants (CNVs) and de novo variants. TDT analysis identified a highly significant locus at 20p12.3, localized to the intergenic region between BMP2 and the noncoding RNA gene LINC01428. Three variants (rs6054763, rs6054764, rs932517) were identified as potential causal variants due to their probability of being transcription factor binding sites, deleterious combined annotation dependent depletion scores, and high minor allele enrichment in probands. Morphometric analysis of cranial vault shape in an unaffected cohort validated the effect of these three single nucleotide variants (SNVs) on dolichocephaly. No genome-wide significant rare variants, de novo loci, or CNVs were identified. Future efforts to identify risk variants for sNCS should include sequencing of larger and more diverse population samples and increased omics analyses, such as RNA-seq and ATAC-seq. “
Sage Science Products:
PippinHT was used to size select whole genome libraries for Oxford Nanopore Promethion sequencing.
Methods Excerpt:
“…3–5 µg of genomic DNA was sheared using a Megaruptor 3 (Diagenode) and purified using Ampure XP beads. Sheared DNA was size selected using the PippinHT instrument (Sage Science) with a target range of 16–20 kb fragments. Next, 1 µg of fragmented, purified, and size-selected DNA in a volume of 47 µl was used in the SQK-LSK109 library preparation protocol per manufacturer’s instructions (Oxford Nanopore Technologies). DNA was end-repaired using the NEBNext FFPE DNA Repair Mix and NEBNext Ultra II End Repair/dA-tailing modules, followed by purification with AMPure XP beads (1:1 vol ratio) and elution to a final volume of 60 µl. Adapters were ligated, and the final library resuspended in Long Fragment Buffer (Oxford Nanopore Technologies). The resulting final library yield was 1.2–2.2 µg per specimen. Libraries were loaded onto PromethION Flowcells (R9.4.1) with 20 femtomolar (fM) loading. After 24 h, all specimens were nuclease washed and reloaded with 20 fM of library. Total sequencing run time was 72 h.”
Author Affiliations:
Statistical Genetics Section, Computational and Statistical Genomics Branch, National Human Genome Research Institute, National Institutes of Health (NIH), Baltimore, MD
Neurobehavioral Clinical Research Section, Social and Behavioral Research Branch, National Human Genome Research Institute, National Institutes of Health (NIH), Bethesda, MD
Center for Craniofacial and Dental Genetics, Department of Oral and Craniofacial Sciences, School of Dental Medicine, University of Pittsburgh, Pittsburgh, PA
Department of Human Genetics, KU Leuven, Leuven, Belgium
Department of Electrical Engineering, ESAT-PSI, KU Leuven, Leuven, Belgium
Medical Imaging Research Center, University Hospitals Leuven, Leuven, Belgium
Department of Pediatrics, University of California Davis, Sacramento, CA
Department of Human Genetics, School of Public Health, University of Pittsburgh, Pittsburgh, PA
Bioinformatics Core, Genome Center, University of California Davis, Davis, CA
Department of Otolaryngology, Head and Neck Surgery, University of California Davis, Sacramento, CA
Department of Neurosurgery, University of California Davis, Sacramento, CA
Pediatric Clinic, Alexandrovska University Hospital, Medical University of Sofia, 1431, Sofia, Bulgaria
Molecular Medicine Center, Department of Medical Chemistry and Biochemistry, Medical Faculty, Medical University of Sofia, 1431, Sofia, Bulgaria
Neuroscience Research Australia, University of New South Wales, Sydney, Australia
Department of Life Sciences and Public Health, Università Cattolica del Sacro Cuore, 00168, Rome, Italy
Fondazione Policlinico Universitario A. Gemelli, IRCCS, 00168, Rome, Italy
Seattle Children’s Craniofacial Center, Center of Developmental Biology and Regenerative Medicine and Division of Craniofacial Medicine, Department of Pediatrics, University of Washington, Seattle, WA
Department of Epidemiology, College of Public Health, The University of Iowa, Iowa City, IA
Nature Scientific Reports
DOI:10.1038/s41598-024-58343-w
Mature microRNA-binding protein QKI promotes microRNA-mediated gene silencing
February 2024
Authors:
Kyung-Won Min, Myung Hyun Jo, Minseok Song, Ji Won Lee, Min Ji Shim, Kyungmin Kim, Hyun Bong Park,Shinwon Ha, Hyejin Mun, Ahsan Polash, Markus Hafner, Jung-Hyun Cho, Dongsan Kim, Ji-Hoon Jeong, Seungbeom Ko, Sungchul Hohng, Sung-Ung Kang & Je-Hyun Yoon
Abstract:
“Although Argonaute (AGO) proteins have been the focus of microRNA (miRNA) studies, we observed AGO-free mature miRNAs directly interacting with RNA-binding proteins, implying the sophisticated nature of fine-tuning gene regulation by miRNAs. To investigate microRNA-binding proteins (miRBPs) globally, we analyzed PAR-CLIP data sets to identify RBP quaking (QKI) as a novel miRBP for let-7b. Potential existence of AGO-free miRNAs were further verified by measuring miRNA levels in genetically engineered AGO-depleted human and mouse cells. We have shown that QKI regulates miRNA-mediated gene silencing at multiple steps, and collectively serves as an auxiliary factor empowering AGO2/let-7b-mediated gene silencing. Depletion of QKI decreases interaction of AGO2 with let-7b and target mRNA, consequently controlling target mRNA decay. This finding indicates that QKI is a complementary factor in miRNA-mediated mRNA decay. QKI, however, also suppresses the dissociation of let-7b from AGO2, and slows the assembly of AGO2/miRNA/target mRNA complexes at the single-molecule level. We also revealed that QKI overexpression suppresses cMYC expression at post-transcriptional level, and decreases proliferation and migration of HeLa cells, demonstrating that QKI is a tumour suppressor gene by in part augmenting let-7b activity. Our data show that QKI is a new type of RBP implicated in the versatile regulation of miRNA-mediated gene silencing.“
Sage Science Products:
PippinHT was used to isolate micro RNA libraries.
Methods Excerpt:
“Library preparation was performed using the TruSeq Small RNA library preparation kit (Illumina, RS-200) following the manufacturer’s instructions. Briefly, 1 μg of input RNA was loaded into Urea-TBE gels for purification and the resulting RNAs were applied for the following procedures. User-supplied reagents including T4 RNA ligase2 Deletion Mutant (Lucigen, LR2D1132K) and Maxima First Stand cDNA synthesis kit (Thermo Fisher Scientific, K1641) were purchased separately. Libraries were amplified using 11 cycles of PCR for the manufacture’s index or modified index primer set to increase diversity. Libraries were prepared according to the manufacturer’s protocol with a modification in the size selection step, which, instead of agarose gel purification, PippinHT Prep instrument (Sage Science, HTP0001) and 3% agarose dye-free cassette with internal standards (Sage Science, HTG3010) was used under the following conditions: base pair start = 120 bp, base pair end = 160 bp, range = broad, target peak size = 145 bp. Eluted Libraries from PippinHT system were subsequently analysed on Tape-station 4150 (Agilent Technologies, G2992AA) following the manufacturer’s instructions using a High Sensitivity DNA Screen tape (Agilent Technologies, 5067–5584). Each library was barcoded with unique sequence of reverse primers during the PCR step, which contained Illumina compatible indices and modified indexes (see GEO database). Before pooling libraries for the next-generation sequencing, concentration of each library was measured in a high sensitivity Tape-station followed by smear analysis. Illumina NextSeq 550 or Miniseq with single end (50 nt;R1) read method was apply for the library sequencing.”
Author Affiliations:
Department of Biochemistry and Molecular Biology, Medical University of South Carolina, Charleston, SC
Department of Biology,Gangneung-Wonju National University, Gangneung, Republic of Korea
Department of Physics & Astronomy, Seoul National University, Seoul,Republic of Korea
Neuroregeneration and Stem Cell Programs, Institute for Cell Engineering, Johns Hopkins University School of Medicine, Baltimore, MD
Department of Oncology Science, University of Oklahoma, Oklahoma City, OK
Laboratory of Muscle Stem Cells and GeneRegulation, National Institute of Arthritis and Musculoskeletal and Skin Diseases, National Institutes of Health, Bethesda, MD
Department of Biochemistry and Molecular Biology, University of Ulsan College of Medicine, Seoul, Korea
RNA Biology
DOI: 10.1080/15476286.2024.2314846