Roy Sooknanan

Abstract 4232: Increased sensitivity in whole-genome bisulfite sequencing (WGBS): A novel “post-bisulfite conversion” library construction method from sub-nanogram DNA inputs.

Genome-wide analysis of 5-methylcytosines is possible with whole-genome bisulfite sequencing (WGBS), where unmethylated cytosine residues are converted to uracil. However, a major challenge in WGBS is the degradation of DNA that occurs during bisulfite conversion under conditions required for complete conversion. Typically, ~90% of input DNA is degraded and thus, is especially problematic with limited starting amounts of DNA. Additionally, regions that are rich in unmethylated cytosines are more sensitive to strand breaks. As a consequence, a majority of DNA fragments contained in di-tagged NGS DNA libraries treated with bisulfite “post-library construction” can be rendered inactive due to strand breaks in the DNA sequence flanked by the adapter sequences. These mono-tagged templates are then excluded during library enrichment resulting in incomplete coverage and bias when performing whole genome bisulfite sequencing. Here, we describe a novel “post-bisulfite conversion” library construction method for preparing NGS libraries from genomic DNA prior to the addition of one or both adapters. This “post-bisulfite conversion” library construction method uses the resulting untagged or mono-tagged single-stranded DNA as template for the subsequent addition of adapter sequences required for NGS. Thus, single-stranded DNA fragments independent of size and position of strand breaks remain as viable templates for library construction, eliminating the loss of fragments and the selection bias associated with a “post-library construction” bisulfite conversion strategy. This novel “post-bisulfite conversion” library construction method exhibits high diversity, increased efficiency and sensitivity (500 picograms human genomic DNA input), and improved coverage required for WGBS.

Abstract 3185: Rapid and efficient methods for preparing rRNA-depleted and directional RNA-Seq libraries from low-input and FFPE RNA samples

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Massively parallel sequencing of cDNA libraries (RNA-Seq) is rapidly becoming the preferred method for transcript profiling, and analysis of novel transcripts, novel isoforms, alternative splice sites, rare transcripts and cSNPs, compared to microarrays. Preparing NGS RNA-Seq libraries typically require first isolating rRNA-depleted RNA or enriching for poly(A)+ mRNA. However, total RNA isolated from formalin-fixed, paraffin-embedded (FFPE) patient tissue samples is normally fragmented, making it not suitable for rRNA-depletion using some commercially available kits or poly(A)+ enrichment, which results in a 3′ sequence bias. Here, we present an efficient “single-pass” rRNA-depletion method (Ribo-Zero™ technology) for use with as little as 100 ng total RNA input from either intact or fragmented (e.g., FFPE) RNA samples. Additionally, an improved, more user-friendly version of the ScriptSeq™ RNA-Seq method (ScriptSeq™ v2) is used to rapidly prepare directional (∼99% strandedness) RNA-Seq libraries in about 2.5 hours, in a single-tube workflow, from either the intact or fragmented Ribo-Zero™ treated RNA samples. The ScriptSeq™ method does not require end-polishing, adaptor-ligation, cDNA fragmentation, or gel-size selection. The combined Ribo-Zero™ and ScriptSeq™ workflow is completed in about 5 hours, generating cluster-ready NGS libraries that contain <2 % of reads that map to rRNA sequences (nuclear- and mitochondrial-encoded) and ∼98% of reads that map to the genome, while maintaining the representation of coding and non-coding transcripts, independent of polyadenylation. This reduction in rRNA sequence reads improves sequence depth and coverage of mRNA, and increases the percentage of uniquely mapped reads required for transcriptome analysis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3185. doi:1538-7445.AM2012-3185

Abstract 4857: Improved technology for ribosomal RNA (rRNA) removal from formalin-fixed paraffin-embedded (FFPE) total RNA

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Deep, massively parallel sequencing of cDNA generated from RNA (“RNA-Seq”) is rapidly gaining momentum for transcript profiling, discovery of novel transcripts, and identification of alternative splicing events. Current methods for making sequencer-specific di-tagged DNA fragment libraries for RNA-Seq typically comprise first, preparing rRNA-depleted RNA from total RNA samples that are usually of good quality followed by the synthesis of the di-tagged cDNA sequencing templates. In the past few years however, it has also become evident from microarray and qPCR studies that formalin-fixed paraffin-embedded (FFPE) cancer tissues hold valuable secrets about diseased states. However, RNA-Seq libraries prepared from such highly fragmented FFPE RNA cancer samples yield limited information since these libraries contain a majority of rRNA reads, which decreases sequencing depth and coverage. Current commercially available rRNA-removal kits are not designed to remove fragments of rRNA, which poses a significant limitation for preparing highly informative RNA-Seq libraries from FFPE RNA samples. Here, we present RNA-Seq results obtained using a novel rRNA removal technology (“Ribo-ZERO™”) and a novel, ligation-free process for preparing directional di-tagged DNA fragment libraries (“ScriptSeq™ Technology”) for RNA-Seq. Using these methods, directional di-tagged DNA fragment libraries can be prepared in about 6 hours from either intact or fragmented (e.g., FFPE) total RNA samples (as little as 100 ng FFPE total RNA sample required). Less than 2 % of the sequence reads from libraries generated from total RNA from either intact or FFPE samples map to rRNA sequences (28S, 18S, 5.8S and 5S). This reduction in rRNA sequence reads from FFPE RNA samples improves sequence depth and coverage, and increases the percentage of uniquely mapped reads, increasing the information obtained from these diseased samples. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4857. doi:10.1158/1538-7445.AM2011-4857