Decontamination of MDA Reagents for Single Cell Whole Genome Amplification

Single cell genomics is a powerful and increasingly popular tool for studying the genetic make-up of uncultured microbes. A key challenge for successful single cell sequencing and analysis is the removal of exogenous DNA from whole genome amplification reagents. We found that UV irradiation of the multiple displacement amplification (MDA) reagents, including the Phi29 polymerase and random hexamer primers, effectively eliminates the amplification of contaminating DNA. The methodology is quick, simple, and highly effective, thus significantly improving whole genome amplification from single cells

Finishing the euchromatic sequence of the human genome

The sequence of the human genome encodes the genetic instructions for human physiology, as well as rich information about human evolution. In 2001, the International Human Genome Sequencing Consortium reported a draft sequence of the euchromatic portion of the human genome. Since then, the international collaboration has worked to convert this draft into a genome sequence with high accuracy and nearly complete coverage. Here, we report the result of this finishing process. The current genome sequence (Build 35) contains 2.85 billion nucleotides interrupted by only 341 gaps. It covers ∼99% of the euchromatic genome and is accurate to an error rate of ∼1 event per 100,000 bases. Many of the remaining euchromatic gaps are associated with segmental duplications and will require focused work with new methods. The near-complete sequence, the first for a vertebrate, greatly improves the precision of biological analyses of the human genome including studies of gene number, birth and death. Notably, the human enome seems to encode only 20,000-25,000 protein-coding genes. The genome sequence reported here should serve as a firm foundation for biomedical research in the decades ahead

CRISPR Explained: Gene Editing History, Technology, and Applications

This video from Bio-Rad Explorer describes CRISPR. In the video, Damon Tighe highlights CRISPR's applications, history, ethics, and activities; describes how CRISPR-Cas9 works; and provides an example of modeling a Cas9 molecular mechanism. The video recording runs 24:02 minutes in length

CRISPR Explained: Gene Editing History, Technology, and Applications

This webinar, published by InnovATEBIO, is the first in a webinar series that presents on CRISPR gene editing. In the video, Leigh Brown, Aaron Kallas, and Damon Tighe, who are scientists at the Bio-Rad Laboratories, discuss the discovery of the CRISPR-Cas9 system in nature, explore how CISPR-Cas9 works in a gene editing scenario, and consider the broad range of applications that this technology possesses. The presentation explores de-extinction projects, designer babies, neo-germ line cancer treatment, and more examples of gene editing. The webinar recording runs 1:28:51 minutes in length

Overcoming some of the challenges to single cell genomics

Single cell genomics, the amplification and sequencing of genomes from single cells, can provide a glimpse into the genetic make-up and thus life style of the vast majority of uncultured microbial cells, making it an immensely powerful and increasingly popular tool. This is accomplished by use of multiple displacement amplification (MDA), which can generate billions of copies of a single bacterial genome producing microgram-range DNA required for shotgun sequencing. Here, we would like to address several challenges inherent in such a sensitive method and propose solutions for the improved recovery of single cell genomes. While DNA-free reagents for the amplification of a single cell genome are a prerequisite for successful single cell sequencing and analysis, DNA contamination has been detected in various reagents, which poses a considerable challenge. Our study demonstrates the effect of UV radiation in efficient elimination of exogenous contaminant DNA found in MDA reagents, while maintaining Phi29 activity. Second, MDA is subject to amplification bias, resulting in uneven and sometimes insufficient sequence coverage across the genome. In a post-amplification method, we employed a normalization step within 454 Titanium library construction in which populations of highly abundant sequences were specifically targeted and degraded from the library via duplex-specific nuclease, resulting in decreased variability in genome coverage. While additional challenges in single cell genomics remain to be resolved, the two proposed methodologies are relatively quick and simple and we believe that their application will be of high value for future single cell sequencing projects

The Joining of Competitors: The Dual Operation of the ABI 3730xl and GE MegaBACE4500 DNA Sequence Analyzers at the DOE Joint Genome Institute

The Joining of Competitors: The Dual Operation of the ABI 3730xl & GE MegaBACE4500 DNA Sequence Analyzers at the DOE Joint Genome InstituteChristopher Daum, Damon Tighe, Lena Philips, Danielle Mihalkanin, Cailyn Spurrell, Don Miller, Alex Copeland, Susan M. Lucas, JGI Sequencing Team U.S. DOE Joint Genome Institute, Walnut Creek, CA 94598At the center of the Department of Energy s (DOE) Joint Genome Institute (JGI) Production Genomics Facility (PGF), lies a highly efficient and automated production line devoted to the generation of high-quality genomic DNA sequence. The JGI utilizes a dual platform of DNA sequence analyzers: Applied Biosystems 3730xl and GE Healthcare s MegaBACE 4500. The operation of these high-throughput fluorescence-based DNA sequence analyzers at the JGI will be assessed on the strengths and benefits of each platform, instrument overviews of operation parameters and mechanical/component specifications. In addition, instrument setups for production operation, operation schedules, loading, and maintenance strategies as well as the various sequencing strategies for each platform. Throughput numbers and sequencing quality results will be presented. UCRL ABS-217110 LBNL-59101 Abs