Targeted sequencing RNA-Seq

Genomic Services

www.ogt.com/genefficiency

Whole exome | Pre-designed panels | Custom panels | RNA-Seq

Delivering comprehensive, high-quality next generation

sequencing services including advanced data analysis

Sequencing Applications

Delivering the complete NGS solution…

Genefficiency sequencing services are designed to be different,

leading you all the way from project conception to high-quality

results (Figure 1). Our expert and comprehensive solutions are

tailored to your specific needs, allowing you to discover new

biological knowledge without being reliant on local

bioinformatics support.

Genefficiency Sequencing Services for DNA and RNA deliver:

• Optimisation of the market-leading technologies to provide

the highest quality results

• A comprehensive interactive data report enabling rapid access

to meaningful results without the need for in-house

bioinformatics resource

• Expert advice on experimental design

• A dedicated project manager to ensure delivery of high-quality

results in the agreed timeframe and budget

Figure 1. The comprehensive, high-quality Genefficiency Sequencing Services workflow.

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• Expert advice on

experimental design,

capture and analysis

• Dedicated project

manager

• Quality control

monitoring and

reporting throughout

the process

• Full QC metrics

integrated into data

report

• Use of OGT optimised

market-leading

technologies

• Successful delivery of

large genomic projects

to time and on budget

• Optimised, robust

analysis pipelines

• Stringent QC metrics

to ensure meaningful

results

• Bespoke analysis

available

• Interactive report

giving access to

meaningful results

without local

bioinformatics resource

• No additional software

required

Experimental design Array design & format Sample processing Analysis Report

Oxford Gene Technology (OGT) is a

leading genomic services provider. It

was founded by Professor Ed

Southern, with offices in Europe and

the US. Our purpose-built, state-of-

the-art facilities are combined with

experienced multi-disciplined teams

and quality systems to deliver projects

on time, within budget and to the

highest standards.

…from sample to result

Effective experimental design

Each sequencing project is assigned a dedicated lab and bioinformatics project

manager. An initial project consultation is provided as standard to discuss:

• Aims of the project

• Nature of the samples

• Platform and technology to be used

• Most appropriate analysis pipeline to address the specific project aims

• Demonstration of the interactive report

• Timeframe of the project

Powerful interactive report

OGT has developed a user friendly, unique and complimentary software package that allows users to rapidly

identify relevant results — overcoming the common bottleneck of handling the huge NGS data sets. Through

an easy-to-use, mouse-click navigation system, thousands of variants can be filtered within minutes to just a

handful for further validation (Figure 2). For RNA sequencing, the navigation system allows rapid access to

differentially expressed transcripts (Figure 3).

Dedication to quality

OGT offers a comprehensive, flexible, high-throughput sequencing service for projects ranging from 1 to 1000s

of samples. Industry-leading platforms, chemistries, automated workflows and an advanced Laboratory

Information Management System allow us to employ stringent quality control metrics at every stage of the

process, giving you the highest confidence in the results delivered. The OGT management system is certified to

the ISO9001:2008 and ISO27001:2005 international standards for quality and information security

management by the British Standards Institute (BSI).

Figure 2: Partial filtering of mutations to identify the

causative mutation in a rare disease.

Figure 3: Visualise the difference in transcript

expression levels influencing gene expression.

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Whole exome sequencing and…

Whole exome sequencing

Whole exome sequencing focuses on the 1.5% of the human

genome corresponding to the gene encoding regions that contain

approximately 85% of disease causing mutations1. As such, it is

significantly faster and more cost-effective than whole genome

sequencing. Screening the protein coding variants against variant

databases allows rapid identification of previously unreported

genetic mutations and potential implications of previously

characterised mutations.

The OGT Whole Exome Sequencing Service includes a variety of

analysis options matched to the aims of each individual project,

such as familial studies, rare disease2 and cancer sequencing.

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“Using OGT’s sequencing and analysis

expertise we were able to filter over

100,000 DNA variants to a single

causative mutation in a matter of

minutes. We were delighted with the

speed and quality of service, plus the

ease-of-use of the analysis platform.”

Dr Bernd Wollnik,

Institute of Human Genetics,

University Hospital of Cologne

• Individual sample

• Variants called against a reference genome

• Report developed for standard pipeline

• Multi-sample comparisons

• Paired sample comparisons for cancer studies

• Family cohort analysis

• Rare disease analysis

• Variants called by sample and across samples

• Bespoke analysis to meet specific project requirements

Standard Analysis

Expert Analysis

Advanced Analysis

Typical protocol:

Agilent SureSelect™

(V4 or V5) capture

products and

sequencing on an

Illumina® HiSeq™

2000

Discover the power of the Genefficiency NGS report

Simple click navigation allows rapid identification of relevant results.

• Sort variants by genotype

• Identify novel variants

• Filter for predicted protein effect and consequence

• Rapid visual inspection of variants through IGV

Custom bait design for targeted re-sequencing

Targeted panel sequencing allows you to look at smaller panels of genes relevant to a specific disease or a

chromosomal region of interest. It offers the benefits of more cost-effective sequencing, flexibility in choice of

regions to be targeted and improved coverage of regions of interest, which may be missed or under-

represented in exome capture. Efficiency and uniformity of capture are critical to successful variant detection.

Non-uniform or poorly enriched regions can result in missed variants.

To improve the likelihood of variant detection right across your regions of interest, we offer an expert bait

design pipeline, which delivers better capture performance than Agilent eArray™ (Figure 4).

OGT’s custom panel designs offer:

• Increased uniformity and efficiency of

capture

• Decreased off-target noise

• Increased sensitivity for variant detection

• Improved capture of GC-rich targets

…custom targeted re-sequencing

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Figure 4. OGT’s expert bait design delivers improved efficiency

and uniformity of target sequence capture. A 25-gene custom

panel was designed using standard software (SSD1-8) and the

OGT optimised design algorithm (OGT1-8). With OGT expert

design, the percentage of bases sequenced to at least 0.2x

mean coverage increased from 73% to >91%. A further design

optimisation subsequently improved this to >95%.

Additional filtering options include:

• Depth of coverage

• Specific gene lists

• Allelic frequency

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RNA-Seq for differential gene expression analysis…

The OGT RNA-Seq Service delivers high-quality mRNA and whole transcriptome sequencing and comprehensive

analysis. RNA-Seq is most commonly used to investigate differential gene expression of known transcripts (Figure 5)

but this can be accompanied by much broader discoveries at the transcriptome level. For example, variant detection,

alternative splicing patterns (Figure 6), identification of fusion proteins and isoform identifications can be carried

out with confidence at the correct sequencing coverage. At OGT, we offer guidance on the coverage required

whether you are looking for array-equivalent expression data, quantification of known or novel splicing variants

or identification of novel transcripts and for discovery projects focusing on non-coding RNAs.

Delivery of an intuitive report

The complimentary RNA-Seq report utilises a simple mouse-click navigation system, enabling quick interrogation

of data to deliver meaningful results without the requirement for additional local bioinformatics support.

Discover the power of the Genefficiency NGS report

Rapid access to differential gene expression and structural variant information.

• Identify changes in expression levels of specific genes

• Compare transcript and gene expression levels

• Analyse splice variants

• Results on known genes

• Gene and transcript levels of expression

• Promoter and coding sequence differences

• Identification of indels

• Pathway analysis (GO/GSEA)

Standard differential gene expression plus:

• De novo transcript discovery and annotation

• Alternative splicing

• Fusion protein

• SNPs

• Bespoke analysis to meet specific project requirements

• Advanced structural variation studies

• Time course studies

Differential GeneExpressionAnalysis

Expert WholeTranscriptomeAnalysis

WholeTranscriptomeAnalysis

Typical protocol:

NEBNext® Ultra™

Directional RNA library

preparation and

sequencing on an

Illumina® HiSeq® 2000

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…and structural variation analysis

OGT’s RNA-Seq Service has been used to investigate changes in gene expression during the development of

drug resistance, the change in splicing after disruption of regulatory proteins and allele specific expression.

Find out more about OGT’s RNA-Seq services at www.ogt.com/rna-seq.

• See the quality score linked to the differential expression data for

confidence in results

• Smoothly switch from global view of gene expression to examine

specific genes or transcripts

• Easy access to nucleotide-level detail or ontology information

Figure 5. Rapid visualisation of differentially

expressed transcripts contributing to

differential gene expression.

Figure 6. With a simple mouse click

transition to analysis of the splice variants of

transcripts across the gene compared to the

reference sequence.

Sample handling expertiseProjects have been successfully completed on

DNA and RNA from cell lines, fresh tissue and

formalin-fixed, paraffin-embedded (FFPE) samples.

Within applications such as whole exome

sequencing and targeted re-sequencing, OGT’s

highly-skilled molecular biologists have vast

experience of sample handling and experimental

design — ensuring your precious samples are in

safe hands. As part of our involvement in a

Technology Strategy Board funded Solid Tumour

Profiling for Stratified Medicines project, we have

optimised the sequencing workflow utilising FFPE

samples allowing you to unlock the potential in your sample archive. Good concordance between matched FFPE

and fresh samples has been achieved even when starting with as little as 50-500ng of genomic DNA (Figure 7).

For more informationFind out more about OGT’s Genefficiency Genomic Services at www.ogt.com/genefficiency or contact

us at services@ogt.com to discuss your project and to request a quote.

References

1. Choi, M. et al (2010) Genetic diagnosis by whole exome capture and massively parallel DNA sequencing. Proc. Natl. Acad. Sci. USA

106, 19096-19101

2. Netzer, C. et al (2012) A mutation in the 5’-UTR of IFITM5 creates an in-frame start codon and causes autosomal-dominant

osteogenesis imperfect type V with hyperplastic callus. Am. J. Hum. Genet. 91(2):349-57

For research use only. Not for use in diagnostic procedures.

This document and its contents are © Oxford Gene Technology IP Limited – 2013. All rights reserved. OGT™ and Genefficiency™ are trademarks of Oxford Gene

Technology IP Limited. SureSelect™ and eArray™ are trademarks of Agilent Technologies. HiSeq™ and MiSeq™ are trademarks of Illumina Inc.

Oxford Gene Technology

Begbroke Science Park, Begbroke Hill, Woodstock Road, Begbroke, Oxfordshire, OX5 1PF

T: +44 (0) 1865 856826 (US: 914-467-5285) E: services@ogt.com www.ogt.com

LungTissue

Sample 1 Sample 2

9 8

27

27 shared variants9 3

28

28 shared variants

Figure 7. Good concordance between matched FFPE and

fresh samples. Targeted sequencing of 2 pairs of matched

fixed (FFPE; light blue) and fresh (yellow) lung samples

using a 25-gene cancer panel indicated high level

concordance between detected variants.