Concurrent Workshop (Genomics): GRG (Genomics Research Group) / DSRG (DNA Sequencing Research Group)
Single-cell Genomics: Best practices and new insights.
Single-cell RNA sequencing (scRNA-Seq) offers great new opportunities for increasing our understanding of complex biological processes. In particular, development of an accurate Human Cell Atlas is largely dependent on the rapidly advancing technologies and molecular chemistries employed in scRNA-Seq. These advances have already allowed an increase in throughput for scRNA-Seq from 96 to 80,000 cells on a single instrument run by capturing cells within nano-liter droplets. In addition, use of oligonucleotide labeled antibodies in combination with scRNA-Seq techniques (e.g. CITE-Seq, Total-Seq, REAP-Seq) allows for multiplexing and to pair transcriptome information with cell surface protein expression to enhance the value of these data as well as to allow for novel insights into the complex biology. Use of such “barcoded” antibodies approached has immense value in understanding complex tissues and developmental processes, such as lung and hematopoietic development, as well as understanding the intricate composition of cancer tissue at the cellular level to help inform treatment strategies. Here we present some initial findings on best practices for use of barcoded antibodies with 10X Genomics technology and provided some data on the added value that can be extracted by using cell surface protein markers to help gain insights into complex tissue organization.
Cross Site Evaluation of Sanger Sequencing Dye Chemistries
Sanger sequencing remains an essential tool utilized by researchers and despite competition from commercial providers, many sequencing core facilities continue to offer Sanger sequencing services to their customer base. By reducing costs and providing rapid turnaround times, in-house Sanger sequencing remains a viable core service, often helping to subsidize more costly services such as next generation sequencing. While Applied Biosystems’ BigDye ™ Terminator chemistry was once the only solution available for Sanger DNA sequencing, several new products employing novel dye chemistries and reaction configurations have entered the market. Currently, it is unclear how these new chemistries perform on various DNA templates, including difficult templates or their amenability to commonly employed cost-saving measures such as dye dilution and reaction miniaturization. With this goal in mind, we compared the quality of Sanger sequencing data produced by kits available from several vendors using control and difficult-to-sequence DNA templates under various reaction conditions. This study will serve as a valuable resource to core facilities conducting Sanger sequencing, providing guidelines on appropriate protocols to use with each kit and determining the most cost effective solutions for Sanger sequencing while maintaining high quality results.