Transcript The BCM Microarray Core Facility

The BCM Microarray Core Facility: Closing the
Next Generation Gap
1
Raza ,
1
Hoang ,
1
Silva ,
1
Liles ,
1
Broadbent ,
1
Mou ,
1,2
White
Alina
Mylinh
Gayan De
Laura
Carl
Yanglong
Lisa D.
1Microarray Core Facility and Dept. Molecular & Human Genetics, 2Dept. Molecular & Cellular Biology,
Baylor College of Medicine, Houston, TX USA
INTRODUCTION
The Microarray Core Facility (MCF) at Baylor College
of Medicine provides investigators with access to a
variety
of state-of-the-art
technologies
and
approaches that will enhance discovery for their
genomic research. We house instrumentation
supporting Affymetrix, Agilent, NimbleGen, Luminex,
and Illumina platforms. The MCF provides expertise
in the following applications: gene expression
profiling using arrays, array comparative genomic
hybridization (aCGH), SNP genotyping, and nextgeneration sequencing applications including RNAseq, ChIP-seq, Targeted genomic resequencing,
miRNA-seq, and de novo sequencing. In addition,
our lab offer services for sample quality check and
provides access to a 150K cDNA clone repository, for
those that are interested in verifying results from
gene expression experiments or any other
application requiring cDNA clones.
NEXT-GENERATION SEQUENCING
APPLICATIONS AVAILABLE
Sample Quality
oAgilent Bioanalyzer
oBio-Rad Experion
oNanoDrop
oGel Electrophoresis
Gene Exp. Profiling
omRNA
Affymetrix
Agilent
NimbleGen
SuperArray
Luminex/Panomics
qPCR
omicroRNA
Luminex FlexmiR
Agilent
Affymetrix
qPCR
New
oExon/alternative splicing
Affymetrix
qPCR
DNA Analyses
oSNP Genotyping
Affymetrix SNP 6.0
Luminex xTAG
qPCR
oChIP-on-Chip/epigen.
Agilent
Affymetrix
Nimblegen
oaCGH
Agilent
NimbleGen
Affymetrix SNP 6.0
Clone Repository
Data Analysis
Illumina Genome Analyzer IIx sequencing
LIBRARY QUALITY:
• Gene expression and alternative splicing (mRNA-Seq)
• Protein-nucleic acid association profiling/epigenetics (ChIP-Seq)
• Sequencing targeted genomic regions (DNA-seq)
• Small RNA discovery (small RNA-Seq)
• De novo genomic DNA sequencing
• 36, 50, or 75 cycle, single-end or paired end
PAIRED-END SEQUENCING
AGILENT BIOANALYZER
The combination of read-length and paired-end flexibility allows for high quality
genomic sequencing applications. Illumina’s sequencing chemistry supports 100+ bp
single-reads, short- and long-insert paired-end reads for whole-genome sequencing
and resequencing, SNP discovery, identification of copy number variations and
chromosomal rearrangements.
RESOURCES
The MCF is a full service facility offering exemplary processing
in applications ranging from transcriptomic profiling to
specialized DNA analyses. Using Affymetrix, Agilent, and
Nimblegen array technology we enable researchers to monitor
genome-wide expression profiles and answer questions about
gene regulation including investigations of alternative splicing
and miRNA profiling. Our mid-range throughput technology
utilizes the Luminex Bead System to measure multiplexed (330 genes) gene expression in a 96-well plate format. We also
offer QPCR analyses utilizing the SA Biosciences SuperArray
platform for gene expression profiling. Chromosomal copy
number changes and CNV analyses of genomic DNA are
performed using Agilent’s aCGH/CNV and Affymetrix SNP
Arrays.
QUALITY CONTROL
After the first read, the clusters are modified to regenerate the template for the paired read.
SAMPLE WORKFLOW for mRNA-seq
1-10 mg of Total
RNA
Isolate mRNA and
Fragment
1st and 2nd strand
cDNA synthesis
BIO-RAD EXPERION
Extensive QC steps are undertaken throughout sample processing. Total
RNA quality is initially checked using the Agilent Bioanalyzer. Genomic
DNA, PCR fragments, and cDNA are checked using either 2% agarose
gel electrophoresis or the Agilent Bioanalyzer.
Internal sample
processing QC steps include testing library size and yield using either
the Agilent Bioanalyzer or the Bio-Rad Experion instrument. Library size
must be checked before proceeding to the Cluster Station to ensure
appropriate clusters will grow on the flowcell. The two images above
illustrate our comparison of the Agilent Bioanalyzer and the Bio-Rad
Experion resulting in essentially no differences. Both instruments appear
to work equally well at measuring size, quality, and concentration of the
sequencing libraries.
LIBRARY QUANTIFICATION:
qPCR is used to quantify libraries to
generate the accurate number of
reads on the sequencer. Samples
are measured against a control of
known concentration, which is run
with the samples on every flowcell.
CONCLUSIONS
T
A
A
T
Ligate Adapters
Size selection and
gel purification
Using Illumina GenomeStudio software to analyzer mRNA-seq reads, sequence tables are generated providing
information on the position of the sequence within the chromosome. Information on SNPs present in a given
sample are also provided in a table describing the position of the SNP, the reference base and the base called.
Enriched cDNA Library by
PCR
Cluster Generation on
Flow Cell
Sequencing on GAIIx
 Illumina’s fast and cost-effective technology gives researches tremendous
amounts of high-quality data to analyze in many applications
 Applications include de novo sequencing, resequencing of whole genomes
and target DNA regions (single and paired end reads)
Diabetes and Endocrinology
Research Center