Dimitrios G Anastasakis, Alexis Jacob, Parthena Konstantinidou, Kazuyuki Meguro, Duncan Claypool, Pavol Cekan, Astrid D Haase, Markus Hafner
The authors offer an improvement to the PAR-CLIP method that is more streamlined and does not require the use of radioactive labels. CLIP-seq (CrossLinking and ImmunoPrecipitation) is used to evaluate protein-RNA interactions during gene expression. PAR-CLIP uses in vivo labelling of RNA with photoreactive nucleosides . The method outlined in this protocol (fPAR-CLIP) uses direct ligation of fluorescent adapters to RNA/protein complexes, followed by the isolation of the RNA. The original PAR-CLIP method requires size fractionation on denaturing polyacrylamide gels. The fPAR-CLIP method presented here eliminates this requirement, is more sensitive, and is completed in 2 rather than 4 days.
Pippin Prep is used to size select PCR fragments and eliminate primers and adapters.
Laboratory of Muscle Stem Cells and Gene Regulation, National Institute for Arthritis and Musculoskeletal and Skin Disease, NIH, Bethesda MD
Laboratory of Cellular and Molecular Biology, National Institutes of Diabetes and Digestive and Kidney Diseases, NIH, Bethesda MD
Laboratory of Clinical Immunology & Microbiology, National Institute of Allergy and Infectious Diseases, NIH, Bethesda MD
MultiplexDX, Bratislava, Slovakia
Nucleic Acids Research
Ruijiao Xin, Yan Gao, Yuan Gao, Robert Wang, Kathryn E. Kadash-Edmondson, Bo Liu, Yadong Wang, Lan Lin & Yi Xing
The authors present a protocol, isoCirc, for the full-length sequence determination of circular RNAs. The method features rolling circle amplification followed by nanopore sequencing, and includes a description of an integrated computation pipeline. A catalogue of over 100,000 circular RNAs across 12 human tissues and the HEK293 cell line was produced, including ~40,000 isoforms.
BluePippin was used to size select the RCA products after debranching prior to ONT MinION library prep.
Center for Computational and Genomic Medicine, The Children’s Hospital of Philadelphia, PA
Genomics and Computational Biology Graduate Program, University of Pennsylvania
Department of Computer Science and Technology, Center for Bioinformatics, Harbin Institute of Technology, Harbin, China
Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, PA
Tao Zhu, Keyan Liao, Rongfang Zhou, Chunjiao Xia & Weibo Xie
The authors suggest an improvement on the ATAC-seq method (Assay for Transposase-Accessible Chromatin with high-throughput sequencing). By using unique molecular identifiers to distinguish between transposase insertions and PCR duplicates. They go on to demonstrate the UMI-ATAC-seq method more accurately quantifies chromatin accessibility and improve the sensitivity of identifying transcription footprints.
The PippinHT was used to size select libraries prior to sequencing.
Huazhong Agricultural University, Wuhan China
Nature Communications Biology
Complete and haplotype-specific sequence assembly of segmental duplication-mediated genome rearrangements using targeted CRISPR-targeted ultra-long read sequencing (CTLR-Seq)
Bo Zhou, GiWon Shin, Stephanie U. Greer, Lisanne Vervoort, Yiling Huang, Reenal Pattni, Marcus Ho, Wing H. Wong, Joris R. Vermeesch, Hanlee P. Ji, Alexander E. Urban
In this preprint the authors report on a method, CTLR-Seq, that combines Sage Science’s HLS-CATCH with low input Oxford Nanopore sequencing(using the MinION platform). The researchers are studying complex and highly repetitive genomic regions associated with neuropsychiatric disorders (mutations at 22q11.2 and at 16p11.2). Long read nanopore sequencing is used to resolve large segmental duplications, copy number variations, and large deletions.
Stanford University, Stanford CA
KU Leuven, Flanders Belgium
Ultra-low input single tube linked-read library method enables short-read second-generation sequencing systems to generate highly accurate and economical long-range sequencing information routinely generate highly accurate and economical long-range sequencing information
Zhoutao Chen, Long Pham, Tsai-Chin Wu, Guoya Mo, Yu Xia, Peter L Chang, Devin Porter, Tan Phan, Huu Che, Hao Tran, Vikas Bansal, Justin Shaffer, Pedro Belda-Ferre, Gregory Humphrey, Rob Knight, Pavel Pevzner, Son Pham, Yong Wang and Ming Lei
This study describes the TELL-Seq™ barcode linked read method developed by Universal Sequencing Technology Corp, and presents validation data for the process. TELL-Seq uses a transposase enzyme method that is linked to a bead (TELL-Bead) to insert an 18 bp molecular barcode into illumina sequencing libraries. The method allows economical analysis of haplotype phasing and structural variation in genomes or metagenomic analyses. The workflow requires low DNA input (0.1-5 ng) and is completed in a single tube.
Universal Sequencing Technology, Canton MA
Bioturing, Inc., San Diego CA
University of California, San Diego