Amplification-free long read sequencing reveals unforeseen CRISPR-Cas9 off-target activity
February 2020
Authors:
Ida Höijer, Josefin Johansson, Sanna Gudmundsson, Chen-Shan Chin, Ignas Bunikis, Susana Häggqvist, Anastasia Emmanouilidou, Maria Wilbe, Marcel den Hoed, Marie-Louise Bondeson, Lars Feuk, Ulf Gyllensten, Adam Ameur
Info:
In this preprint, the authors propose a method for screening for off off-target CRISPR/Cas9 sites using PacBio and Oxford Nanopore sequencing. The amplification-free method purifies and sequences both on-target and off-target cut sites from a DNA sample. The method could prove useful for improving gRNA design and screening and suggests the ability to develop de Novo guides as opposed to requiring a reference genome. SageELF was used to fractionate (1-18KB) SMRTBells for PacBio Hi-Fi circular consensus sequencing.
Author Affiliations:
Uppsala University, Sweden
Broad Institute of Massachusetts Institute of Technology and Harvard, Cambridge, MA
Boston Children’s Hospital, Boston, MA
Monash University, Melbourne, Australia
Citation:
bioRxiv
DOI: 10.1101/2020.02.09.940486
Capture of complete ciliate chromosomes in single sequencing reads reveals widespread chromosome isoforms
December 2019
Authors:
Kelsi A. Lindblad, Jananan S. Pathmanathan, Sandrine Moreira, John R. Bracht, Robert P. Sebra, Elizabeth R. Hutton & Laura F. Landweber
Info:
Researchers sequenced the whole genome of Oxytricha trifallax, a model protozoan ciliate, using long read sequencing. Since Oxytricha trifallax has 16,000 chromosomes that only average 3.2 kpb in length, its much of the genome was sequenced with chromosomal-length single reads without assembly. By comparing the results to the reference sequence assemblies the researchers demonstrated that short read sequencing can mask considerable structural variation.
BluePippin was used to size-select PacBio SMRT libraries.
Author Affiliations:
Columbia University, New York, NY
Princeton University, Princeton, NJ
American University, Washington DC
Icahn School o f Medicine at Mt Sinai, New York, NY
Cold Spring Harbor Laboratories, Cold Spring Harbor, NY
Citation:
BMC Genomics
DOI: 10.1186/s12864-019-6189-9
Comparison of mitochondrial DNA variants detection using short- and long-read sequencing
August 2019
Authors:
Ahmed N. Alkanaq, Kohei Hamanaka, Futoshi Sekiguchi, Masataka Taguri, Atsushi Takata, Noriko Miyake, Satoko Miyatake, Takeshi Mizuguchi, Naomichi Matsumot
Info:
The genotypes of mitochondrial DNA variants are compared using both short-read (Illumina Hi-SeqX10) and long-read (PacBio Sequel) sequencing. DNA samples from three individuals with mitochondrial disease used for the study. The authors conclude that although long reads have higher error rates (though random) and short-read have higher accuracy (but with systemic errors), PacBio sequencing can overcome is error bias with 37 reads.
BluePippin was used to select >10kb SMRTbell libraries using the High-Pass protocol.
Author Affiliations:
Yokohama City University
Citation:
Journal of Human Genetics
DOI: 10.1038/s10038-019-0654-9
Ingestible Osmotic Pill for In‐vivo Sampling of Gut Microbiome
July 2019
Authors:
Hojatollah Rezaei Nejad, Bruno C. M. Oliveira, Aydin Sadeqi, Amin Dehkharghani, Ivanela Kondova Jan A. M. Langermans, Jeffrey S. Guasto, Saul Tzipori, Giovanni Widmer, Sameer R. Sonkusale
Info:
From Engineering and Nano Labs at Tufts University, and a consortium of Veterinary groups from the Netherlands, Brazil, and Tufts. A 3D printed micro-engineered pill with osmotic pumping technology was developed to sample the gut microbiome in animals. The pill was coated to resist exposure to the stomach microbiome and can be magnetically held in the upper intestinal track until sampling is complete.
The system was validated in vitro and in vivo. The Pippin Prep was used to size select 16S amplicons prior to Illumina sequencing.
Citation:
Advanced Intelligent Systems
DOI: 10.1002/aisy.201900053
Distinct Profiles of Cell-Free MicroRNAs in Plasma of Veterans with Post-Traumatic Stress Disorder
July 2019
Authors:
Min Young Lee, David Baxter, Kelsey Scherler , Taek-Kyun Kim, Xiaogang Wu , Duna Abu-Amara, Janine Flory, Rachel Yehuda, Charles Marma, Marti Jett, Inyoul Lee, Kai Wang, and Leroy Hood
Info:
Researchers report on studies that profile circulating miRNAs in combat veterans suffering from Post Traumatic Stress Disorder (PTSD). Previous studies had shown that circulating miRNA spectra in extra cellular vesicles (EV) in patients suffering from psychiatric disorders. The authors confirm that miRNA profiles differ between PTSD and non-PTSD patients in both EV and EV-depleted fractions of plasma. Further study is recommended to understand the utility of these findings.
PippinHT was used to isolate modified Illumina small RNA sequencing libraries (134-162bp size selection for 20-22 bp inserts). The method (Giraldez et al. 2019) uses custom degenerate adapters (4N adapters) and increased polyethylene glycol (PEG) during the ligation process. This reduces bias and optimizes several other parameters to make it appropriate for use specifically with plasma RNA.
Author Affiliations:
Institute for Systems Biology, Seattle
Steven and Alexandra Cohen Veterans Center for Posttraumatic Stress and Traumatic Brain Injury, New York University, New York
Icahn School of Medicine at Mount Sinai, New York
James J. Peters VA Medical Center, New Yourk
Integrative Systems Biology, US Army Center for Environmental Health Research, Frederick, MD
Citation:
Journal of Clinical Medicine
DOI: 10.3390/jcm8070963