Genome Editing (GERG)

Investigating FFPE conditions on downstream nucleic acid assays
There are three specific aims in this study. Aim 1).Creating a set of FFPE samples representative of common variations in clinical processing and storage conditions. Aim 2). Evaluating the quality of nuclei acids of these FFPE samples by DIN/RIN value, A260/280, etc at three storage time points, such as one month, six months and twelve months. Aim 3). Further assessing nuclei acid quality of these FFPE samples through gDNA/RNA amplifiability by qPCR and artifactual mutation occurring during FFPE tissue preparation by NGS library sequencing.

The specific aim 1 in the study B has been finished.

Hybrid Capture Methods for Targeted RNA sequencing
This study aims to evaluate how five commercial exome capture kits perform in a targeted RNA-seq application.  Specific Aims : evaluating intra- and inter- site reproducibility of commercially available hybrid capture kits for RNAseq; characterizing absolute quantitative accuracy or limitations of targeted RNAseq; evaluating this method for relative differential gene expression analysis in intact and degraded RNA materials; providing a practical comparison of current capture-based targeted RNAseq protocols.

Cross Site Evaluation of Sanger Sequencing Dye Chemistries

Specific Aims:

1. Evaluate equivalence of routine and difficult Sanger Sequencing templates using a variety of legacy and new-to-market reagents.
2. Provide guidance to ABRF Sanger Sequencing facilities with regard to feasibility of modifying existing dye terminator chemistry protocols to further cost-savings potential.

This study is finished. Jessica W. Podnar gave a presentation at ABRF 2019 annual meeting ( March 23-26, San Antonio, TX).

For more information, please check https://abrf.org/sites/default/files/abrf_sanger_final_2019.pdf

The manuscript is in preparation.

Small RNA Illumina Libraries
This study is finished. Dr. Stuart Levine gave a presentation at ABRF 2018 annual meeting ( April 22-25 Myrtle Beach, SC). For more information, please check dsrg18_presentatin.pdf.pdf 

A poster related to this study was presented at AGBT 2019 by Zachary T. Herbert. 

https://abrf.org/sites/default/files/agbt_smrna_poster_final.pdf

The manuscript is submitted.

RNAseq Illumina Libraries using Ribosomal Depletion - PHASE I

Ribosomal RNA (rRNA) comprises at least 90% of total RNA extracted from mammalian tissue or cell line samples. Informative transcriptional profiling using massively parallel sequencing technologies requires either enrichment of mature poly-adenylated transcripts or targeted depletion of the rRNA fraction. The latter method is of particular interest because it is compatible with degraded samples such as those extracted from FFPE and also captures transcripts that are not poly-adenylated such as some non-coding RNAs. Here we provide a cross-site study that evaluates the performance of ribosomal RNA removal kits from Illumina, Takara/Clontech, Kapa Biosystems, Lexogen, New England Biolabs and Qiagen on intact and degraded RNA samples. We find that all of the kits are capable of performing significant ribosomal depletion, though there are differences in their ease of use. Most kits perform well on both intact and degraded samples and all identify ~14,000 protein coding genes from the Universal Human Reference RNA sample at >1FPKM. A manuscript is currently under preparation. This project has been completed. The collected data were published https://doi.org/10.1186/s12864-018-4585-1 .  

 - View Presentation about progress, 2017 [pdf]

The immediate goal of the study is to objectively assess the technical performance of different next-generation sequencing technologies for RNA analyses, and to evaluate the pros and cons of various data analysis methods. The ultimate goal is to create a publicly available tool for assessment of data from these instruments as compared to an established set of sequencing data from a standardized protocol. The study was initially organized in 2011, and is a joint study with GVRG, NARG, and MARG. Currently the study is being done entirely with current members of the Research groups listed above.

Cross-Site Comparison of Enzymatic Illumina Library Construction Kits 
The ability to produce genomic DNA libraries for Illumina sequencing depends on fragmentation of the DNA.   Fragmentation of high molecular weight DNA has typically been accomplished through physical means such as nebulization or sonication.  These techniques are not readily scalable for high throughput production.  In response, various manufacturers have developed strategies for enzymatic fragmentation of the DNA.  An open question is how well these techniques provide unbiased representation of the samples under study, and whether nucleotide composition of the target material plays a role. To address this question the DNA sequencing Research Group (DSRG) of the Association of Biomedical Research Facilities (ABRF) compared four different methods for enzymatic fragmentation of DNA prior to next-gen library generation.  Both human genomic DNA and bacterial pooled DNAs representing a spectrum of GC compositions were assessed. Kits were analyzed for their resulting library complexity as well as bias in sequence as well as genomic context as well as their site-to-site consistency. These results will have implications for the selection of kits for specific experimental contexts.

A Snap Shot into the Life Cycle of Sequencing in Genomics Core Facilities

The past couple years have seen a dramatic shift in the genomics core facility environment. The rapid evolution of next-generation sequencers, the increasing number of commercial and institutional service providers, and significant shifts in funding, have created a challenging environment for core facilities to keep current with the services and instrumentation they offer.  The ABRF DNA Sequencing Research Group (DSRG) conducted a web-based survey to gain insight into the current status of sequencing core facilities that offer Sanger or Next Generation Sequencing (NGS) services.  Since the introduction of high throughput DNA sequencing by Roche 454 in 2005, there have been a number of platforms available including the current market leader, Illumina, as well as contenders like the ABI 5500xl SOLiD and Ion Torrent platforms; and the third generation PacBio RS system.  This survey focused on current instrumentation, utilization and shifts between instruments as technologies have evolved.  We have also quantified core involvement in surrounding methodologies, such as Sanger sequencing, NGS library preparation and bioinformatics support allowing us to compare investigator needs, scientific applications and technologies across facilities.  

Inter-Laboratory Comparisons of DNA and RNA Quality Assessment

Quantifying RNA and DNA samples is one of the most basic methods used in all sequencing and microarray labs.  A broad spectrum of instruments and techniques are used to ascertain nucleic acid quantity and quality, in preparation for more sophisticated analyses.  Each laboratory tends to favor specific methods.  However, beyond the manufacturer’s specifications, there are few standards to indicate how the instruments are performing relative to their optimal behavior, nor are there easy methods to directly compare platforms to examine relative benefits versus costs.  We have surveyed over 40 core facilities to identify what equipment is currently being used.  We have also directly measured the inter-lab reproducibility and inter-platform reproducibility of different DNA/RNA analyzers as well as their dynamic range using both RNA and DNA standards.  We find that there is significant interlab performance variability of the systems.  Test samples sent to fourteen laboratories showed large variation in quantification of samples and in the dynamic range of the instruments.  We have collected the protocols of the best performing labs on each instrument and present them here as a resource to users to help them achieve optimum performance of their quality control processes.

Metagenomics
A Study of various methodologies used in metagenomic experiments performed on Next Generation DNA sequencing systems. The methodologies include DNA extraction/purification, various platforms, and varying analysis methods. This study is ongoing with an expected completion in 2012, and being done in its entirety by members of DSRG.
- View Presentation about progress, 2011 [pdf]

DNA Capture, Phase II --- Comparison of Commercially Available Target Enrichment Methods for Next Generation Sequencing with the Illumina Platform : Phase II - Solution Capture Methods.

This study is complete with an expected publication date of 2012.
- View Presentation on Progress, 2011 [pdf]