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

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 25-30 years until the development of NGS Genome analyszer in 2005 TP53 c.818G>A p.Arg273His Tumour protein p53 (tumour suppressor)

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.

Overview of the NGS Service in Cardiff NGS has been up and running in IMG since 2012. 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

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

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.

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

Indexing HaloPlex Nextera/ TruSight

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

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: £565.07 per sample TruSight Increase the diversity of the library, increases the cluster density. Viral genome completes with sample for producing clusters.

Cluster Generation on the Miseq Reference: Student Guide: TruSight Rapid Capture with MiSeq. Illumina University. Part # 15049537 Rev. A

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

Sequencing by Synthesis 2nd Read Sequencing read 2 Sequencing by Synthesis 2nd Read

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

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

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.

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)