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NEXT GENERATION SEQUENCING SERVICE GUIDE


ABOUT SOURCE BIOSCIENCE

Source BioScience is an international genetic analysis and diagnostics business

serving the research and healthcare markets. The group headquarters are based in

Nottingham, U.K. operating state of the art laboratory facilities with additional

laboratories in Oxford, Cambridge, Dublin, Rochdale and Germany. Source

BioScience provides a comprehensive portfolio of genetic analysis services and

diagnostic testing’s for both the Research and Healthcare sectors.

Life Science Research

Source BioScience LifeSciences is the European leader in DNA sequencing and

genomic services. We offer a diverse range of services and products, including

Sanger Sequencing, Next Generation Sequencing, Genotyping, Gene Expression,

Bioinformatics, Clones and Antibodies. Our extensive portfolio includes over 20

million clones and over 100,000 antibodies, plus a wide range of research reagents

for applications such as cell culture, immunology and nucleic acid analysis.

Further information about our Services and Products can be found on our

website: www.lifesciences.sourcebioscience.com

As a leading provider of genomic research service in the UK, Source BioScience

is pleased to offer our customers Next Generation Sequencing using the

Illumina HiSeq 2000 and MiSeq platforms.

Our Key Strengths are:

Over a decade of experience in the sequencing field

We have the largest commercial capacity for Illumina Sequencing in the UK with two

HiSeq 2000 and one MiSeq platforms, providing our clients with the fastest

turnaround times.

Dedication to providing the highest quality service to our clients

Our service is certified to the Illumina sequencing CSPro™ status and operates a

robust quality management system. In addition, we are also GLP, GCP and CPA

certified, ensuring that our clients enjoy the highest quality of data and service.


Individual project managers for every project

We understand the importance of your project. An individual project manager is

assigned to your project and will be your point of contact. They will continually

update you on the progress of your project throughout the process.

Advanced Bioinformatic Solutions

We offer advanced bioinformatics solutions for every application to assist

researchers in the analysis of the vast amounts of data produced by the Illumina

Sequencing Systems.

This guide explains our Illumina sequencing service and processes which we hope

will help you in understanding our service.

Contact Us

We are happy to talk to you about experimental designs and to assist you in grant

applications. If you have any queries regarding our Illumina sequencing service, please do

not hesitate to get in touch.

Tel (UK): 0800 652 6774 Tel (Non UK): +44 (0) 115 973 9018 Fax: +44 (0) 115 973 9021 Email: [email protected]


SEQUENCING PLATFORMS

INTRODUCTION

The Illumina HiSeq 2000 and MiSeq are groundbreaking platforms for genetic

analysis and functional genomics. Both platforms combine’s Illumina’s Sequencing-

by-Synthesis (SBS) technology providing high sequencing throughput and fast data

generation. The HiSeq 2000 generates up to 300Gb (gigabase) of data per

sequencing run, equivalent to 37.5Gb per lane. The MiSeq generates up to 13.2-

15Gb of data per sequencing run. The Illumina sequencing platforms combines

innovative engineering with advanced SBS chemistry technology to provide high

outputs, simplicity and cost-effectiveness making any sequencing project possible.

NGS SEQUENCING PLATFORMS PROVIDED BY SOURCE BIOSCIENCE

1. HiSeq 2000

2. MiSeq

SEQUENCING PLATFORM: HISEQ 2000

INTRODUCTION TO HISEQ 2000

The Illumina HiSeq 2000 utilizes Illumina’s reversible terminator sequencing by

synthesis (SBS) chemistry with cutting-edge scanning and imaging technology. The

HiSeq 2000 is able to dramatically increase the number of reads, sequence output

and data generation rate. The HiSeq 2000 has the ability to operate a single or dual

flow cell mode offering experimental flexibility. The flow cells are operated

independently of each other to allow different read lengths to be run

simultaneously.

FIGURE 1: ILLUMINA HISEQ 2000, IMAGE PROVIDED BY ILLUMINA


SEQUENCING ON THE HISEQ 2000

After Library Preparation, the libraries are ready to be sequenced. To prepare the

libraries for sequencing, the libraries are amplified by cluster generation using the

cBot. The cBot is a revolutionary automated clonal amplification system providing

efficiency and ease of use for the highest quality sequencing results. The process of

clonal amplification on the cBot is called Bridge Amplification. Bridge amplification

produces clusters that are used for sequencing.

CLUSTER GENERATION

Cluster generation is completed on the cBot. The cBot uses Bridge Amplification to

create hundreds of millions of single-molecule DNA templates. The Illumina Flow

Cell is coated in oligonucleotides complimentary to the adapters used to generate

the libraries. The oligonucleotides enable the adapters on the DNA strands to bind

to the surface of the Illumina Flow cell. There are 8 lanes on an Illumina Flow cell,

allowing one sample to be run per lane or more if multiplexed.

FIGURE 2: CLUSTER GENERATION BY BRIDGE AMPLIFICATION


CLUSTER GENERATION STEP-BY-STEP

TEMPLATES CAPTURED BY OLIGONUCLEOTIDE LAWN ON FLOW CELL

DNA LIBRARY DILUTED & DENATURED

TEMPLATES BOUND TO THE PRIMERS ARE 3’ EXTENDED, PRODUCING COVALENTLY

ATTACHED DISCRETE SINGLE MOLECULE

DOUBLE STRANDED MOLECULE IS DENATURED AND ORGINAL TEMPLATE WASHED AWAY

FREE ENDS OF BOUND TEMPLATES HYBRIDZE TO ADJACENT LAWN PRIMERS TO FORM

U-SHAPED BRIDGES

THE DNA BRIDGE IS COPIED FROM THE PRIMER TO CREATE A DOUBLE STRANDED DNA

BRIDGE

THE RESULTING DOUBLE STRANDED DNA IS DENATURED FROM A HYBRIDIZED TO LAWN-

PRIMERS TO FORM NEW BRIDGES AND EXTEND AGAIN

REPEAT 35 TIMES TO CREATE A DENSE CLUSTER OF OVER 2000 MOLECULES

REVERSE STRANDS IN THE CLUSTER ARE REMOVED BY CLEAVAGE ST THE REVERSE

STRAND SPECIFIC LAWN PRIMERS, LEAVING A CLUSTER WITH FORWARD STRANDS ONLY

THE FREE 3’ OH ENDS ARE BLOCKED TO PREVENT NON-SPECIFIC PRIMING

SEQUENCING PRIMERS ARE HYBRIDIZED TO FREE ENDS OF THE DNA TEMPLATES


SEQUENCING BY SYNTHESIS (SBS)

The clustered flow cell is ready to be sequenced using the SBS chemistry on the

HiSeq 2000. Hundreds of millions of DNA fragment clusters are sequenced in

parallel using a propriety reversible-terminator method that detects single

nucleotide base as they are incorporated into growing DNA strands. The SBS

chemistry uses four fluorescently labeled deoxynucleotide triphosphate (dNTP) to

sequence the clusters present on the flow cell. Each nucleotide label serves as a

terminator for polymerization, thus after each dNTP incorporation, the fluorescent

dye is imaged to identify the base and then enzymatically cleaved to allow

incorporation of the next nucleotide. The process of adding the dNTP, scanning and

imaging of the base occurs at each cycle until the DNA strand is sequenced. All four

reversible terminator-bound dNTPs (A, G, T, C) are present as single, separate

molecules, natural competition minimizes incorporation bias. The base calls are

made directly from the signal intensity measurements during each sequencing

cycle. This results in an accurate base–by-base sequencing that greatly reduces

sequence-context specific errors enabling robust base calling across the genome,

including repetitive regions.

FIGURE 3: ILLUSTRATION OF SEQUENCING BY SYNTHESIS (SBS)


HISEQ 2000 READ LENGTH AND DATA OUTPUT

The sequencing read length and data output for the HiSeq 2000 can be summarized

in the table below.

FIGURE 4: TABLE OF HISEQ 2000 SYSTEM PERFORMANCE, READ LENGTHS AND DATA

OUTPUT

SEQUENCING PLATFORM: MISEQ

INTRODUCTION TO THE MISEQ

The Illumina MiSeq is the only next-generation sequencer to integrate amplification,

sequencing and data analysis into one single instrument. With the same SBS

chemistry as the HiSeq, the MiSeq is able to provide a rapid turnaround time and

cost-effective genetic analysis for the widest range of applications. The rapid

turnaround time of the MiSeq makes it a desirable instrument for sequencing as

data can be returned within as little as four hours.


FIGURE 5: ILLUMINA M ISEQ, IMAGE PROVIDED BY ILLUMINA

SEQUENCING ON THE MISEQ

The all-in-one MiSeq platform incorporates cluster generation, paired-end fluidics

and completes data analysis. The MiSeq is able to perform cluster generation

without the need of another instrument, sequencing is completed using SBS

chemistry followed by data analysis. Primary analysis is performed by the MiSeq

which includes base calling, filtering and quality scoring. Secondary analysis is

performed to include alignment and variant calling. The primary and secondary

analysis is performed using Illumina MiSeq Reporter software. Customers are able

to use Illumina’s BaseSpace, the next-generation sequencing cloud computing

environment which enables researchers to easily and securely analyze, archive and

share MiSeq data.

The Illumina MiSeq is the first sequencer to be integrated into a cloud platform

allowing data to be pushed to the cloud for automatic storage and analysis. Source

Bioscience customers can create a BaseSpace account and this would enable us to

share the MiSeq data files with the customer through a collaborator site. This allows

access to NGS data on any internet enabled device including tablets and smart

phones.

BaseSpace also allows MiSeq user access to a multitude of sequence analysis apps

allowing applications ranging from genome visualization to de-novo assembly of

small genomes and variant calling.

Further information on BaseSpace can be found on the Illumina Website:

https://basespace.illumina.com/home/index


MISEQ READ LENGTH AND DATA OUTPUT

The sequencing read length and data output for the MiSeq is summarized in the

table below.

FIGURE 6: TABLE OF MISEQ SYSTEM PERFORMANCE , READ LENGTH AND DATA OUTPUT

PLATFORM COMPARISON

This quick guide is to give an overview of the two Next-Generation Sequencing

platforms we have to offer, the HiSeq 2000 and MiSeq. The table gives a

comparison of both platforms and what applications the platforms can be used for.

HISEQ 2000 MISEQ


HiSeq 2000 MiSeq

OUTPUT Up to 300 Gb Up to 13.2-15 Gb

SINGLE READS (MAXIMUM)

1.5 billion total 187 million/lane

22-25 million total

PAIRED- END READS

(MAXIMUM)

3 billion 374 million/lane

44-50 million total

REQUIRED INPUT

50ng with Nextera 100ng-1ug with TruSeq

50ng with Nextera 100ng-1ug with TruSeq

READ LENGTH Up to 2 x 100bp Up to 2 x 300bp

PERCENTAGE OF BASES >Q30

>85% (2 x 50bp) >80% (2 x 100bp)

>85% (2 x 75bp) >70% (2 x 300bp)

APPLICATIONS

Whole Genome Sequencing

Exome Sequencing Targeted Resequencing Transcriptome Sequencing

ChIP Sequencing Small RNA Sequencing Amplicon Sequencing

Whole Genome Sequencing Exome Sequencing

Targeted Resequencing Transcriptome Sequencing

ChIP Sequencing Small RNA Sequencing

Metagenomics Sequencing Amplicon Sequencing

APPLICATIONS

Next generation sequencing (NGS) platforms enable a wide variety of applications,

allowing researchers to ask virtually any question of the genome, transcriptome and

epigenome of any organism. There are a range of library preparation kits that offer

protocols for sequencing whole genomes, mRNA, targeted regions such as whole

exomes, custom-selected regions, protein-binding regions and more. The sample

preparation protocols are very straightforward. Researchers start with gDNA or RNA

samples. DNA or cDNA fragments are then ligated to platform-specific


oligonucleotide adapters needed to perform the sequencing biochemistry. The

samples are then clustered onto the flow cell in preparation for sequencing.

The Illumina Sequencing Platforms supports multiple applications including:

1. Whole Genome Sequencing

2. Exome Sequencing

3. Transcriptome Sequencing

4. Small RNA Sequencing

5. Chromatin-Immunoprecipitation Sequencing (ChIP-Seq)

6. Metagenomics Sequencing

7. Targeted Re-Sequencing

Each section will have information about the library preparation kits we use at

Source BioScience. There will be web links to each of the library preparation kits if

you need further information. Each of the library preparations allow for the

multiplexing of large numbers of samples on a single sequencing run. Most of the

standard kits allow for multiplexing of 48 samples per run but some allow up to 96

samples per run.

WHOLE GENOME SEQUENCING

Whole genome sequencing determines the complete DNA sequence of an organism.

This includes sequencing of all of an organism’s chromosomal DNA as well as DNA

contained in the mitochondria or chloroplast. The cost of sequencing a genome has

reduced dramatically as the technology greatly improves. This gives us the

opportunity to discover more about variations in genomes by utilizing these

powerful NGS platforms.

The NGS Platforms are able to offer single and pair-end reads to enable the

discovery and confirmation of mutations, chromosomal rearrangements or single

polynucleotide polymorphisms through re-sequencing or de novo assemblies.

Re-sequencing enables researchers to align the data to a known reference genome,

whereby de novo assembly reads are aligned without a reference genome.

De novo assemblies are challenging as a range of factors can affect the data quality

such as short read lengths and size and continuity of contigs which may present

gaps in the data and reduce alignment. To overcome this issue, pair-end reads

sequence both ends of the DNA fragment which results in superior alignment,

particularly across repetitive sequencing regions and produces longer contigs that


align better and gives a fuller complete coverage. Mate pair sequencing with large

insert also can provide useful information for assembly of repetitive regions in the

genome and results in more complete assemblies. Source Bioscience also offer the

UK largest commercial Sanger sequencing service which can be used to close gaps

after a de novo assembly has been created from NGS data.

The popular areas in which researchers are taking advantage of this powerful

technology are:

• Human & Mammalian Genetics: Detection of known and novel mutations in

specific regions of interest using targeted re-sequencing

• Microbial sequencing: using de novo and re-sequencing of multiple strains

for identification of phenotype specific mutations

• Agrigenomics: using de novo and re-sequencing of species of commercial

and research interest

• Pathogen detection in host systems: Detection and characterization of

foreign DNA in infected samples from plants or animals

For Whole Genome Sequencing, Source BioScience uses the latest library preparation

kits to prepare the samples ready for sequencing. The library preps we currently

use are:

• Illumina TruSeq Nano DNA Library Preparation

http://www.illumina.com/documents/products/datasheets/datasheet_truseq_

nano_dna_sample_prep_kit.pdf

• Illumina TruSeq DNA PCR-Free Library Preparation


dna_pcr_free_sample_prep.pdf

• Illumina Nextera DNA Library Preparation

http://www.illumina.com/documents/products/datasheets/datasheet_nexter

a_dna_sample_prep.pdf

• Illumina Nextera XT Library Preparation


a_xt_dna_sample_prep.pdf

• Illumina Nextera Mate Pair Sample Preparation


a_mate_pair.pdf

Whole genome sequencing techniques can also be applied to the sequencing of

smaller constructs such as plasmids, cosmids, fosmids and BAC’s.


TRANSCRIPTOME SEQUENCING (RNA-SEQ)

The transcriptome is a collection of all transcripts present in a given cell. This

includes various kinds of RNA such as mRNA and rRNA. The Transcriptome

sequencing (also known as RNA-Seq) sequences cDNA to further obtain information

about a sample’s RNA content. This enables researchers to characterize RNA

transcribed from a particular genome to decipher information about gene

regulation and protein function. Stranded information identifies from which of the

two DNA strands a given RNA transcript was derived. This information is useful in

determining strand orientation to identify antisense expression. The ability to

capture sense and antisense expression provides insight into regulatory

interactions that might have been missed. This provides a further understanding of

specific cell types, how a cell functions and changes in gene activity can contribute

to disease.

At Source BioScience we understand the importance of Transcriptome Sequencing

and have an extensive library preparation protocols to assist with your research.

The library preps we currently use are:

• Illumina TruSeq RNA Library Preparation – This gives non-stranded

information after a poly-A purification of mRNA from a total RNA sample.


sample_prep_kits.pdf

• Illumina Stranded Total RNA Library Preparation – This gives stranded

information after a ribo-depletion of rRNA from a total RNA sample.


stranded_rna.pdf

• Illumina Stranded mRNA Library Preparation - This gives stranded

information after a poly-A purification of mRNA from a total RNA sample.


stranded_rna.pdf

• Clontech Smarter Ultra Low RNA Library Preparation – Useful for low RNA

input (10pg-10ng)

http://www.clontech.com/GB/Products/cDNA_Synthesis_and_Library_Constru

ction/cDNA_Synthesis_Kits/Ultra_Low_Input_RNA_cDNA_Synthesis?sitex=100

30:22372:US

• NuGen Ovation RNA-Seq FFPE Library Preparation - Used for Formalin

Fixed Paraffin Embedded samples.

http://www.nugeninc.com/nugen/index.cfm/products/cs/ngs/ovation-rna-

seq-ffpe-system/

• Epicentre Ribo-Zero Library Preparation - These kits remove ribosomal RNA

in samples before proceeding to sequencing library preparation. Depletion


of ribosomal RNA as opposed to poly-A purification of mRNA allows for

sequencing analysis of non-polyadenylated RNA’s such as lincRNA’s.

http://www.epibio.com/products/rna-sequencing/rrna-removal/ribo-zero-

rrna-removal-kits-selection-guide

o We have kits for different species such as:

� Human/Mouse/Rat

� Gram Positive Bacteria

� Gram Negative Bacteria

� Metabacteria

� Plant

� Yeast

o Script-Seq Library Preparation

http://www.epibio.com/products/rna-sequencing/rna-library-

prep/scriptseq-v2-rna-seq-library-preparation-kit?details

SMALL RNA SEQUENCING

Small RNA sequencing is a powerful application to discover the profiling of micro

RNAs and other non-coding RNA on any organism without prior knowledge of the

genome. This would include sequencing micro RNA (miRNA), short interfering RNA

(siRNA) and piwi-interacting RNA (piRNA). Small RNA Sequencing allows researchers

to identify expression patterns, disease association and to characterize previously

unknown small RNAs.

At Source BioScience we use the latest products from Illumina to enable us to

sequence small RNAs with the most accurate detection and quantification of rare

small RNA sequences. The small RNA library prep available to our customers is:

• Illumina TruSeq Small RNA Sample Prep Kit

http://support.illumina.com/documents/documentation/Chemistry_Documentation/SamplePre

ps_TruSeq/TruSeqSmallRNA/TruSeq_SmallRNA_SamplePrep_Guide_15004197_E.pdf

CHROMATIN-IMMUNOPRECIPITATION SEQUENCING (ChIP-SEQ)

Chromatin-Immunoprecipitation sequencing has enabled researchers to identify

protein-DNA interactions and its regulation expressions to understand many

biological processes and disease states. ChIP-Seq is an extremely versatile

application and has successfully been applied against a wide range of protein


targets, including transcription factors and histones. ChIP produces libraries of

target DNA sites bound to the protein of interest in vivo. The ChIP process enriches

specific crosslinked DNA-protein complexes using an antibody against the protein

of interest. Oligonucleotide adapters are then added to the small stretches of DNA

that were bound to the protein of interest. After size selection, the ChIP DNA

fragments are sequenced on the Illumina HiSeq 2000 or MiSeq.

At Source BioScience we offer the latest ChIP library prep for your research needs to

enable high-quality, cost-efficiency and high-throughput data. The ChIP library prep

available to our customers is:

• Illumina TruSeq ChIP Sample Preparation

http://www.illumina.com/documents/products/datasheets/datasheet_truseq_chip_sample_pre

p_kit.pdf

METAGENOMICS SEQUENCING

Metagenomics is the study of metagenomes (all the genetic material present in an

environmental sample, consisting of the genomes of many organisms). This enables

researchers to discover new hidden and diverse microscopic life to enable further

understanding of the living world. Sequence variation in the 16S ribosomal RNA

(rRNA) gene is widely used to characterize taxonomic diversity present in microbial

communities. The 16S sequence is composed of nine hypervariable regions

interspersed with conserved regions. The sequence of the 16S rRNA gene and its

hypervariable regions has been identified in a large number of organisms. For

taxonomic classification it is sufficient to sequence individual hypervariable regions

instead of the entire gene length. In most microbial species the fourth hypervariable

(V4) region is approximately 254bp and only deviates by a few base pairs. The 16S

V4 region is sequenced on the MiSeq to generate high quality, full length reads of

the entire V4 region. This enables microbial profiling at the Species level from the

V4 region of the 16S ribosomal RNA. Our current protocol allows for the

multiplexing of hundreds of samples in a run. Please feel free to contact us if you

need more information.

At Source BioScience, we offer this exciting service for your research needs.

Currently this service is offered on the MiSeq only as it produces high-quality data

and faster turnaround time. The MiSeq Reporter software also enables data analysis

of your samples to identify the variety of metagenomes in your sample. Customers

can be supplied with both classification data as well as sequencing reads derived

from each sample.


We are also in a position to offer sequencing of other regions other than the V4

region mentioned above. Please feel free to contact us with your specific

requirements.

TARGETED RE-SEQUENCING

Targeted Re-sequencing enables researchers to focus on small subset of the

genome such as, an exome, particular chromosome, a set of genes or region of

interest. Focusing on a subset of the genome reduces costs but also allows

researchers to only sequence regions that they are interested in. This technique is a

popular approach to sequence many individuals to discover, screen or validate

genetic variation within a population. This enables researchers to identify rarer

variants that were missed or too expensive to indentify using other sequencing

methods. There are two different approaches to creating libraries for targeted

sequencing and Resequencing projects – target enrichment and amplicon

sequencing.

TARGET ENRICHMENT

Target enrichment captures selected regions or genes enriched in the library from

genomic DNA. Target enrichment allows larger DNA insert sizes and enables a

greater amount of total DNA to be sequenced per sample. This enables researchers

to further expand the information that they can gather from each sample. For

example, rather than sequencing a few hundred exons, the entire exome can be

sequenced to identify functional single nucleotide polymorphisms (SNPs) for an

individual. This application enables researchers to identify rare disease-associated

alleles within a population.

At Source BioScience, we offer a wide range of target enrichment products to aid

customer’s research needs. The target enrichment kits that we used are:

• Illumina Nextera Rapid Capture Exome and Expanded Exome Kits

http://www.illumina.com/documents/products/datasheets/datasheet_nextera_rapid_capture_

exome.pdf

• Illumina Nextera Rapid Capture Custom Enrichment Kit

http://www.illumina.com/documents/products/datasheets/datasheet_nextera_rapid_capture_

custom_enrichment.pdf

• Illumina TruSight Cancer

http://www.illumina.com/documents/products%5Cdatasheets%5Cdatasheet_trusight_cancer.p

df

• Illumina TruSight Cardiomyopathy

http://www.illumina.com/documents/products\datasheets\datasheet_trusight_cardio.pdf

• Illumina TruSight Inherited Disease


http://www.illumina.com/documents/products\datasheets\datasheet_trusight_inherited_disea

se.pdf

• Illumina TruSight Autism

http://www.illumina.com/documents/products%5Cdatasheets%5Cdatasheet_trusight_autism.p

df

• Illumina TruSight Tumor

http://www.illumina.com/documents/products/datasheets/datasheet_trusight_tumor.pdf

• Illumina TruSight One Sequencing Panel

http://www.illumina.com/documents/products/datasheets/datasheet_trusight_one_panel.pdf

• Agilent SureSelect XT Human Exome Enrichment

http://www.chem.agilent.com/library/brochures/SureSelect-HaloPlex-NGS-Brochure-5990-

8747EN.pdf

• Agilent HaloPlex Human Exome Enrichment


8747EN.pdf

• Agilent SureSelect XT Custom Target Enrichment


8747EN.pdf

• Agilent HaloPlex Custom Human Target Enrichment


8747EN.pdf

• Agilent SureSelect XT Mouse Exome Enrichment


8747EN.pdf

• Agilent HaloPlex Cancer Panel

http://www.chem.agilent.com/library/flyers/Public/Haloplex%20Cancer_Flyer_Final.pdf

• Agilent HaloPlex Cardiomyopathy Panel

http://www.chem.agilent.com/library/datasheets/Public/HaloPlexDiseaseResearchPanels5991-

2526ENa.pdf

• Agilent HaloPlex Arrhythmia Panel


2526ENa.pdf

• Agilent HaloPlex Noonan Syndrome Panel


2526ENa.pdf

• Agilent HaloPlex ICCG Panel


2526ENa.pdf

• Agilent HaloPlex Connective Tissue Disorder Panel


2526ENa.pdf

• Agilent HaloPlex X Chromosome Panel


2526ENa.pdf


AMPLICON SEQUENCING

Amplicon sequencing allows researchers to sequence small, selected regions of the

genome spanning hundreds of base pairs. This approach enables researchers to

create thousands of amplicons spanning multiple areas of interest in multiple

samples that can be simultaneously prepared, making it more cost-effective than

previous sequencing methods. Amplicon sequencing has a wide range of

applications for discovering, validating and screening genetic variants for various

studies. Using NGS, sequencing amplicons give a high depth of coverage to identify

common and rare sequence variations. This is useful for the discovery or rare

somatic mutations in complex samples, e.g. cancerous tumors mixed with germline

DNA. This makes amplicon sequencing suitable for clinical environments where

researchers are limited to disease-related variants.

Source BioScience offers a range of amplicon kits to prepare samples for sequencing. The

kits we use are:

• Illumina TruSeq Custom Amplicon

http://www.illumina.com/documents//products/datasheets/datasheet_truseq_custom_amplico

n.pdf

• Illumina TruSeq Cancer Panel

http://www.illumina.com/Documents/products/datasheets/datasheet_truseq_amplicon_cancer

_panel.pdf

Custom sequencing requires the design of custom capture probes or oligos and both

Illumina and Agilent provide online resources to design custom kits.

Illumina:

http://www.illumina.com/applications/designstudio.ilmn

Agilent:

https://earray.chem.agilent.com/suredesign/

SERVICE TYPE

We offer two types of services:

- Full Service

This includes sample library preparation and sequencing run.

- Run Only

This includes sequencing run only.


NGS SEQUENCING WORKFLOW From Sample to Sequence…

This is a general NGS sequencing workflow to show you what happens to the samples when

they reach our lab.

SAMPLES ARRIVE INTO

LAB

SAMPLES ARE QUALITY

CHECKED TO SEE IF

THEY MEET THE SAMPLE

REQUIREMENTS

PASS

CUSTOMER INFORMED & SAMPLES

CONTINUE TO LIBRARY

PREPARTION

FAIL

CUSTOMER INFORMED & NEW

SAMPLES REQUESTED

LIBRARY PREPARATION

& LIBRARY REPORT QC SENT TO

CUSTOMER

CLUSTER GENERATION ON CBOT

(FOR HISEQ RUN ONLY)

SEQUENCING ON HISEQ 2000 OR

MISEQ

BIOINFORMATICS

DATA SENT BACK TO CUSTOMER VIA

FTP (SERVER LINK) OR HARD DRIVE

(HHD) WITH BIOINFORMATICS REPORT


SAMPLE REQUIREMENTS

The input material we require exceeds the amounts required for sample

preparation. This is due to the additional amount that is necessary to perform

initial QC on the samples.

SAMPLE TYPES AND REQUIREMENTS

Library preparation required Input requirements Minimum concentration

Buffer

ChIP seq 20ng – 350bp 1ng/µl TE buffer

Genome sequencing 2µg genomic DNA 100ng/µl TE buffer

Targeted re-sequencing 6µg genomic DNA 100ng/µl TE buffer

mRNA - seq (total RNA) 2µg total RNA 100ng/µl DEPC water

mRNA – seq (mRNA) 200ng mRNA 6.25ng/µl DEPC water

DGE – small RNA (total RNA) 2µg total RNA 500ng/µl DEPC water

Mate pair 2µg genomic DNA 100ng/µl TE buffer

Nextera (Nextera XT) 200ng DNA 10ng/ µl DEPC Water

Run only 100nM 10nM Water

Please contact us if your sample requirements do not meet the minimum requirements

QUALITY CHECK (QC) METHODS

We will perform an initial QC of all sample material as follows:

Library preparation Method Quantification Method

ChIP seq Agilent 2100 High Sensitivity DNA chip

Genome sequencing Qubit Flourometer dsDNA broad range assay

Targeted re-sequencing Qubit Flourometer dsDNA broad range assay

mRNA - seq (total RNA) Agilent 2100 RNA Nano chip

mRNA – seq (mRNA) Agilent 2100 RNA Pico chip

DGE – small RNA (total RNA) Agilent 2100 RNA Nano chip

Mate pair Qubit Flourometer dsDNA broad range assay

Nextera (Nextera XT) Qubit Flourometer dsDNA high sensitivity assay

Run only Agilent 2100 High Sensitivity DNA chip

If the samples received do not meet our QC requirements, we will contact you to discuss

how to proceed.

DNA QUALITY

When submitting DNA we recommend measuring the concentration with

fluorescence based measurements (such as the Qubit). This is preferable to the

Nanodrop which tends to overestimate the concentration of the DNA. A Nanodrop


however is useful for determining the 268:280 ratios of submitted samples. For

highest quality sequencing a range of 1.8:2 is desirable.

The quality of DNA can be assessed by running ~50ng of DNA on a 1% agarose gel.

Good quality DNA should appear as a strong band at a high molecular weight

(>10000bp). The presence of a lower molecular weight smear would indicate DNA

of insufficient quality or RNA contamination.

RNA QUALITY

The RIN (RNA Integrity Number) of all submitted RNA (except for FFPE samples)

should be above 8. This can be measured using the Agilent BioAnalyzer. If this is

not possible, running the samples on a denaturing agarose gel to confirm the

integrity of the rRNA peaks if preferable. All RNA samples will be run on the Agilent

BioAnalyzer and the RINs measured.

FIGURE 7: RNA INTEGRITY VALUES

FIGURE 9: RNA INTEGRITY ON A GEL.

(Image provided by Invitrogen

http://www.invitrogen.com/site/us/en/home/Refere

nces/Ambion-Tech-Support/rna-isolation/tech-

notes/is-your-rna-intact.html)


After the samples are QC’ed, we will generate an Initial QC report stating whether

the samples meet the library preparation requirements. If the samples fail QC, we

will recommend that new samples are to be submitted. If samples pass QC, we will

prepare the samples according to the relevant protocol.

SAMPLE PREPARATION & LIBRARY VALIDATION

SAMPLE PREPARATION FOR SEQUENCING

Each separate application requires specific sample preparation for the sequencing.

The sample library will be prepared according to the protocol recommended for that

particular application. Further information on each of the library preparations for

various NGS applications can be found under the ‘Application’ section of this guide.

There are website links to each of the different library preparations to provide

further information. If you need further assistance with selecting the correct library

preparation for your project, please contact us.

LIBRARY VALIDATION

After completion of the library preparation, the libraries are validated prior to

cluster generation. This is to ensure that library preparation has worked. We use the

Agilent 2100 BioAnalyser to determine fragment size, yield and concentration

according to the protocol used for the library preparation. We aim to obtain over

10nM of each library before proceeding to sequencing.

LIBRARY PREPARATION REPORT

Customers will receive an extensive library preparation report stating which library

preparation was carried out on their samples. We include further information such

as External and internal sample identification, indexes used, library validation

Agilent BioAnalyser traces, Agilent BioAnalyser traces of Pool (if applicable) and

further recommendation if the library preparation has failed. We understand the

importance of information and we strive to always send this report to our customers

prior to any further sequencing work. If the library preparation has failed we will be

in contact to recommend alternatives or further information as to why the library

preparation did not work. If the library preparation has passed, we wait to make

sure you are happy with the results of the library preparation before continuing on

with sequencing. We aim to make sure that our work is of high quality and is

transparent, so that you receive the best quality from us.


CLUSTER GENERATION

During cluster generation, the prepared library/libraries are hybridized to the flow

cell. Each flow cell is coated with oligonucleotides complementary to the adapters

attached onto the ends of each sample. The samples are flushed through the flow

cell and are bound to the oligonucleotide lawn. Once bound, the DNA strands

undergo bridge amplification to create thousands of clusters for each template.

FIGURE 10: DIAGRAM OF HOW CLUSTER GENERATION WORKS


SEQUENCING

Illumina’s Sequencing by Synthesis (SBS) chemistry enables the incorporation of a

single fluorescent nucleotide, followed by high resolution imaging of the entire flow

cell. Each cluster appears as a dot on the image scanned by the camera. Any signal

above the background identifies the physical location of a cluster and the

fluorescent emission identifies which of the four bases (A, G, C, T) was incorporated

at that position. The fluorophore is removed and a new base is added, scanned and

imaged. This process is continued depending on the read length.

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