1. Next Generation Sequencing
Comparison of Enrichment Protocols
Dilruba Meah
Genetic Technologist
University Hospital of Wales

2. Why NGS?
Sanger Sequencing (developed in 1977) was the most widely used method
Sequencing method based on incorporation of chain-terminating dideoxynucleotide triphosphates (ddNTPs) during in vitro replication.
First Human Genome published in Science and Nature in 2001 took 15 years to sequence and costing ~ 3 billion dollars.
Low sample numbers
Screening small numbers of genes/exons
Sanger Seq discovered by Fredrick Sanger and co-workers was the most predominately used method over years until the development of NGS Genome analyszer in 2005
TP53 c.818G>A p.Arg273His Tumour protein p53 (tumour suppressor)

3. Why NGS? NGS was developed to improve:
Cost
Throughput
Sensitivity
NGS was established in 2005 and allowed multiplexed sequencing of both people and genes
Genome Analyzer (2005) could produce 1 gigabase data
Now 45 Human Genomes can be sequenced in a ~ 24 hours at around 1 thousand dollars using the HiSeq X Series producing up to 1.8 terabases of data.

4. Overview of the NGS Service in Cardiff
NGS has been up and running in IMG since Panels include:
Early onset severe Infantile epilepsy – 36 genes
Cortical Brain Malformation (CorMal) – 43 genes
Set up using the HaloPlex Target Enrichment protocol (Agilent) for Illumina Sequencing.
Cancer Panel:
Hereditary breast/ ovarian cancer – BRCA1 and BRCA2
Tuberous Sclerosis – TSC1 and TSC2
Colorectal Cancer:
Lynch syndrome – MLH1 MSH2 MSH6
Familial adematous polyposis – APC
MUTYH associated polyposis – MUTYH
These are set up using the TruSight Cancer Rapid Capture protocol (Illumina).
CRUK: Late stage metastatic non-small cell lung cancer (NSCLC) – 28 genes
Set up using a custom platform: Nextera Rapid Capture Enrichment protocol (Illumina) which supports the analysis of Tumour samples.
Trusight and CRUK followed later in 2014

5. Sequencing Workflow Sample Preparation Cluster Generation Sequencing
Library Validation
Quality
Quantity
Cluster Generation
MiSeq/ HiSeq flow cell
Sequencing
MiSeq/ HiSeq
Alignment &Data Analysis
Primary
Secondary

6. HaloPlex Library Preparation
Fragment target DNA (10 ng/μl) using restriction enzymes . Enzymes are denatured to prevent further digestion.
Hybridise the HaloPlex probe library to the fragmented DNA.
Hybridisation results in gDNA fragment circularisation and incorporation of indexes and Illumina sequencing motifs.
Capture the targeted probe hybrids using magnetic streptavidin coated beads.
Remove the non- circular fragments.
Close circular molecules by ligation reaction and then elute from the magnetic beads.
PCR amplify targeted fragments to produce a sequencing ready, target enriched sample.

7. Nextera/ TruSight Library Preparation
Tagment gDNA
Transposomes fragment gDNA and adds adapters for PCR
Clean up Tagmented DNA
Removes transposomes from tagmented DNA
First PCR Amplification
Amplifies purified tagmented DNA and adds indexes 1 and 2
First PCR Clean up
Removes unwanted products
50 ng Genomic DNA (5 ng/µl)
First Hybridization
Biotinylated capture probes target DNA ROI
First Capture
Streptavadin magnetic beads used to capture Biotinylated DNA.
Washed and then eluted.
Second Hybridization
Additional capture probes target eluted DNA to ensure high specificity of captured regions
Second Capture
Streptavidin beads used to capture probes hybridised to targeted ROI.
Capture Sample Clean up
Sample purification beads used to purify captured library
Second PCR Amplification
Amplifies enriched DNA library
Second PCR Clean up
Purifies enriched library and removes unwanted products
Library Validation

8. Indexing
HaloPlex
Nextera/ TruSight

9. Library validation HaloPlex Nextera/ TruSight
Bioanalyzer-after restriction digest
Bioanalyzer used for checking quality and concentration
Qubit used to check concentration
Bioanalyzer and Qubit used for checking quality and concentration of library

10. Sequencing Nextera HaloPlex TruSight PhiX: 1 % Read length: 75
Cartridge: V3 150
Run length: 12 hours
Cost: £559 + £123 per FISH test
Read length: 150
Cartridge: V2 300
Run length: 24 hours
Cost: £357.18
HaloPlex
PhiX: 5 %
Read length: 200
Cartridge: V3 600
Run length: 48 hours
Cost: £ per sample
TruSight
Increase the diversity of the library, increases the cluster density. Viral genome completes with sample for producing clusters.

11. Cluster Generation on the Miseq
Reference: Student Guide: TruSight Rapid Capture with MiSeq. Illumina University. Part # Rev. A

12. Sequencing by synthesis
Optimizing flow cell clustering determines data quality and final data yield.
Overclustering:
Loss of data quality and data output
Reduced base calls and Q30 scores
Loss of focus
Complete run failure
Underclustering:
Loss of time and money
Complete failure of run
1. Add polymerase and all 4 dNTPs. Further extension is blocked by a terminator
2. Once the single base addition occurs the clusters can be excited by a laser and the colour of the added base
can be imaged and determined
3. Once the first base has been imaged and determined, the fluorescent label is cleaved and terminator
removed, enabling extension
4. The next cycle begins by adding a new pool of polymerase and dNTPs
5. Repeating this process allows us to step-wise determine the sequence of the original template

13. Sequencing by Synthesis 2nd Read
Sequencing read 2
Sequencing by Synthesis 2nd Read

14. Miseq analysis % > Q30 Cluster density (K/mm2)
Clusters passing filter
Yeild %
Aligned % (phiX)

15. Data Analysis HaloPlex NextGENe Autorun tool Variant comparison tool
Alignment to genome
Generates VCF files with all the calls
Variant comparison tool
Filtering/removing common changes (artefacts)
NEW pipeline now set up for automated analysis
Cluster analysis
1st and 2nd Checking using IGV to interprate variants
Gap filling and confirmations of mutations
Still using Sanger Sequencing (48 and 96-capillary 3730 DNA Analyzers)
MLPA (LIS, DCX)
Dosage analysis (quantitative qPCR method  Halotator)
Reporting

16. Data ANalysis TruSight Nextera MiSeq reporter Custom Pipeline
Alignment to genome
Picard and Manta analysis- structural variation and indel calling for germline and somatic
Custom Pipeline
Generating Variant studio files- checking variants called by the MiSeq Reporter
Filtering out common variants in blood and tumour  tumour specific variants
IGV used to interpreting variants
TruSight
Custom pipline
CUTADAPTA- Adaptor trimming and alignment to whole genome
Remove PCR duplicates
GATK tool kit
Variant detection, filtering and removing false positives. VCF file generated.
Confirmations using MLPA and Sanger. Gap filling using Sanger.

17. Acknowledgement Rachel Butler (IMG Head)
Sheila Palmer-Smith (Clinical Scientist)
Hood Mugaalaasi (Clinical Scientist)
Mat Lyon (Bioinformatician)
Hazel Ingram (Head GT)
Rebecca Harris (Lead GT)