Sequencing and Non-coding RNA Program
The Sequencing and Non-coding RNA Program (ncRNA Core) is a centralized shared resource of the Center for Targeted Therapy at MD Anderson Cancer Center. Our mission is to provide MD Anderson community and its network researchers the most comprehensive data efficiently through the utilization of cutting edge technologies and skilled expertise in functional genomics for DNA genotyping and RNA transcriptome profiling, as well the discovery of novel transcripts in mRNA, LncRNA and small non-coding RNAs (ncRNA) as biomarkers in basic and translational cancer research. Our goal is to provide researchers a full service laboratory including sample preparation through the data production with quick turnaround time, quality data, affordable costs and flexibility to meet your research needs
Illumina NextSEQ 500 Next Generation Sequencing: The ncRNA core provides high throughput sequencing on the Illumina NextSeq 500 sequencer, which is capable of producing up to 400 million single reads and 800 Million Paired-End reads per full run, with output data ~19 - 120 gigabases of sequence within run times ranging from 19-29 hours. There are two type of high or mid output sequencing kits support (see Table). The NextSeq 500 is very scalable allowing it to support several DNA and RNA applications including, sequencing of small genomes, targeted genome sequencing such as ChIP-seq, RIPseq, CLIPseq and whole exome sequencing; Stranded mRNA and whole transcriptome and small RNA sequencing, Epigenetic sequencing to profile DNA CpG island methylation modification as well. You will be able to decide the number of samples to be multiplexed for the application based on the interrogating sample complexity with maximum data coverage needed. The core provides full service library preparation, and sample QC. Included in the cost of sequencing is basic bioinformatic analysis through Illumina BaseSpace (SNP/InDel calling, transcript abundance).
NextSeq 500 Sequencing Performance
Illumina NextSeq 500 NGS Data Analysis and Bioinformatic Support
Sequencing raw data processing and downstream analysis are available through the fee-for-services provided by the bioinformatics team of the Genomic Medicine department using the standard pipeline employed for MD Anderson Moon Shot Program projects and internal/external projects. Detailed information is provided in a subsequent section.
PacBio Sequel SMRT Sequencing
The PacBio Sequel Sequencing is a proven third generation Single Molecule,Real-Time (SMRT) technology that provides ultra-long sequencing reads (8-12Kb) with high consensus accuracy (99.9999%), uniform coverage, and simultaneous epigenetic characterization at single-molecule resolution. Sequel SMRT sequencing directly measures individual molecules, using long reads to fully characterize genetic complexity, including rare SNPs, indels, structural variants, haplotypes and phasing. Single molecule resolution allows comprehensive characterization of heterogeneous samples and identification of variation invisible to multi-molecule sequencing technologies. The PacBio Sequel SMRT sequencing system provides extra-long read lengths for de novel that simplify and improve genome assembly without reference. Iso-Seq RNA sequencing that provide long read full length transcripts and isoforms cross the poly(A) tail to the 5′ end without the need of assembly and is ideal for improving annotations in reference genomes, characterizing gene isoforms, alternative splicing and gene fusion events. The PacBio Sequel SMRT sequencing detects single nucleotide modifications in real time, directly measuring the kinetic properties of base additions during the sequencing process. These kinetic measurements present characteristic patterns in response to a variety of base modifications such as 5-methylcytosine, 5-hydroxymethylcytosine, 6-methyladenine, 8-oxoguanine, and more. Sequel SMRT sequencing expects that researchers will use this capability to study a broad range of base modifications at single base resolution.
Pacbio Sequel Services: “Sequencing and ncRNA program core” offers 1) Sequel SMRT bell DNA and RNA library preparation and SMRT sequencing services. The SMRT bell library preparation can be started from either high molecular weight genomic DNA for whole genome assembly and multiplexed PCR amplicon(s) for targeted genome sequencing. Or cDNA SMRT bell library converted from high quality intact total RNA for whole transcriptome sequencing (Iso-Seq method). Click PacBio Sequel SMRT Sequencing for sample requirements or visit our pricing page.
Life Ion Proton System Next Generation Sequencing:
We offer ion Proton semiconductor sequencing technology that apply biochemistry to directly translate chemical information into digital data. Chip-based semiconductor sequencing enables you to scale your runs to your research needs. The Ion PI™ Chip provides you with 80-100 M reads, enabling you to sequence 1–3 human exomes or 1-8 human RNA whole transcriptomes per run. The application for Ion Proton is ideal for exome sequencing, CHIP sequencing and Ion Ampliseq™ targeted DNA panels and Ampliseq™ human transcriptome RNA panels with rapid results, lower cost and easy streamline data analysis. All pre-designed DNA and RNA Ampliseq™ panels can be found https://www.ampliseq.com/. The Core provides sample QC, sample library and sequencing template preparation and sequencing data production. The features of Ion Proton sequencing is listed as https://www.thermofisher.com/order/catalog/product/4476610
Affymetrix GeneChip-Expression Profiling and Genotyping:
Affymetrix has hundreds of GeneChip microarray cross mot of speciese, if not all. We provide full services for all of Affmetrix GeneChip Products for RNA transcriptome expression profiling and SNP genotyping. The full service include performing sample QC, sample processing and target labeling, array hybridization, post-hybridization washing/staining, array scanning and data crunching. Simple data analysis using free download Affymetrix TAC software is also provided as well. Turnaround time is approximately 3-5 business days.
Affymetrix TAC software URL for download:
Custom Array System-Small ncRNA and miRNA Profiling:
Conducted on in-house microarray platforms and has the capacity to profile up to approximately 48 samples per week. The program’s director, Chang-gong Liu, Ph.D., who developed the first-ever ncRNA/miRNA microarray platform. The core has capability to build custom small ncRNA arrays, such as miRNA and tsRNA arrays with proven data recorded in publications.
HTG EdgeSeq-RNA Extraction-free Targeted RNA Sequencing
HTG molecular Diagnostic HTG Edgeseq is a digital quantitation system using S1 nuclease protection assay to profiling targeted panel of genes directly from the most difficult samples of FFPE, sera/plasma, PAXgene, cultured cells, fresh or frozen tissues, without RNA extraction. HTG has several commercial products available in market. Please visit HTG website for the products and their applications as listed https://www.htgmolecular.com
Real-Time PCR Data Validation: All high throughput microarray and next gene sequencing data need a independent technology to validate it. To support gene expression quantitation and the detection of SNPs, we offer Applied Biosystems 7900HT Fast Real-Time PCR System to your data validation with a turnaround time of approximately two-to-three hours.
RNA/DNA Sample Quality and Quantity Check (QC)
High quality data are generated from high quality intact RNA or DNA as starting materials. We QC every single received RNA and DNA sample using Agilent Bioanalyzer 2100 and Thermofisher Scientific Nanodrop™ 1000 spectrometer and give QC results report within an hour back to sample submitter, before processing.