1. Genomic & RNA Profiling Core Facility
Rene Stalnaker, Sukhinder Sandhu, Ornella Tuekam, Md Habibul Islam, Lisa White, Daniel Kraushaar
Introduction
Illumina
Next Generation
Sequencing
NanoString Technologies
nCounter Assay
The Baylor College of Medicine Genomic and RNA Profiling (GARP) core’s mission is to facilitate cutting-edge genomics research by providing state-of-the-art equipment and expert assistance in strategic and experimental planning.
Our services include next-generation sequencing on the Illumina NextSeq 500 and NovaSeq 6000 systems. We accept user-prepped samples and offer in-house library preparation for applications such as mRNA-seq, Total RNA-seq, Limited-Input RNA-seq, microRNA-seq, ChIP-seq, amplicon sequencing, whole-exome capture, whole-genome and whole genome bisulfite sequencing. Our library prep automation system handles up to 96 samples per run and enables us to generate NGS libraries for low- and high-throughput projects with high reproducibility.
The NanoString Technologies nCounter platform allows for highly multiplexed, direct digital counting of up to 800 targets without the use of enzymes. This PCR-free assay accommodates multiple applications and is well-suited for gene expression studies, microRNA profiling and copy number quantification. The nCounter system can handle challenging sample types such as FFPE and low-input samples.
In addition, we offer RNA and DNA sample quality control, DNA shearing on the Covaris LE220 and automated size selection of DNA products with the Pippin HT.
Here, we present data that illustrates the proficiency of the Swift ACCEL-NGS Methyl-Seq Library Prep kit to generate WGBS libraries from genomic DNA samples at inputs as low as ng.
(1) Overnight hybridization - +
(2) Purification, binding, and alignment on nCounter Prep Station
(3) Digital imaging on nCounter Digital Analyzer
Whole Genome Bisulfite Sequencing
Automated Size Selection
Swift ACCEL-NGS Methyl-Seq Library Prep
QC Metrics for Bisulfite-Converted Whole Genome Sequencing
Sage Science Pippin HT
Sample Source
Input: sheared, bisulfite-converted gDNA
TOTAL READS
% READS ALIGNED
GENOME COVERAGE
% DUPLICATE READS
% CpG SITES METHYLATED
BISULFITE CONVERSION RATE
MEDIAN INSERT SIZE (BP)
Non-Methylated Human gDNA (Zyno)
50 ng
300 M
81.70% 
15.2X 
12.4% 
 42.2% - 
210
10 ng
 14.1X
 15.1%
 40.9%  -
203
Methylated Human gDNA (Zymo)
82.05% 
 15.2X
 11.6%
 99.1%
208
 81.30%
 13.5X
 13.8%
201
Coriell (NA12878) Human gDNA
 84.70%
 8.85X
 13.5%
 77.2%
 98%
196
 83.60%
 12.2%
 81.0%
192
Whole Genome Bisulfite Sequencing (WGBS) in low input amounts: 50 ng and 10 ng sheared bisulfite –converted gDNA.
Bisulfite Conversion achieved using the Zymo EZ DNA Methylation-Gold™ Kit
Sample Sources: Non-methylated Human gDNA control (Zymo), Methylated Human gDNA control (Zymo) and Coriell (NA12878) Human gDNA Control
Eliminates manual gel selection.
Reduces error rates.
Ability to collect narrow and wide size ranges ( bp).
DNA/RNA Shearing
Covaris LE220
WGBS Library Products
Legend
* 50ng Coriell (avg 390 bp)
* 10ng Coriell (avg 381 bp)
* 50ng Methylated (avg 392 bp)
* 10ng Methylated (avg 383 bp)
* 50ng Non-Methyl (avg 389 bp)
* 10ng Non-Methyl (avg 385 bp)
The Swift ACCEL-NGS Methyl-Seq Library Prep with Zymo Methylation-Gold kits demonstrate efficiency in bisulfite conversion across 10 ng and 50 ng following analysis from 300M reads per sample. Alignment scores, duplication rates, genome coverage and uniformity metrics assure confidence in data quality. The aligner tool, bsmap, aligned reads to the hg19 genome. Analysis of Lambda gDNA spike-in, allowed for bisulfite conversion efficiency assessment.
Bioinformatic Analyses performed by Sukhinder Sandhu, Ph.D. (Director of Bioinformatics, Swift Biosciences, Inc)
Adaptive focused acoustics (AFA) technology precisely delivers energy to a specific area within a sample tube.
Size range: bp.
Sample Quality
Control Services
Assay
Sample Type
Reported Data
Agilent 2100 Bioanalyzer
RNA, DNA
Sample integrity and size distribution.
NanoDrop
Sample concentration and purity.
Qubit Fluorometer
Sample concentration.
qPCR
Illumina libraries
Sample concentration of functional molecules for Illumina next generation sequencing.
Agarose Gel Electrophoresis
Genomic DNA
Sample integrity.