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Next Generation Sequencing:A technical perspective

Bristol Genetics LaboratoryJoanne Davies

Genetic Technologist

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Objectives

• Summarise the NGS assays currently available at BGL• Look at the general challenges with NGS assays• Highlight technical issues that could be encountered with specific

library preparation methods– SureSelect– HaloPlex – TruSight

• Briefly consider future plans

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Current NGS assaysNGS Panel Genes Library prep

methodClinical relevance

Inherited Peripheral Neuropathies (IPN)

56 SureSelectXT Genes associated with different types of inherited neuropathy are tested to classify diverse phenotypes

Paediatric Cardiomyopathy (PC)

71 SureSelectXT PC has multiple causes and can present in infancy with cardiac failure and sudden death.

Chromosome Breakage Disorders (CBD)

63 SureSelectXT Diagnosis and classification of Fanconi anaemia and related conditions characterized by a defect in DNA repair mechanisms or genomic instability.

Inherited Bone Marrow Failure Syndromes (IBMFS)

45 SureSelectXT Classification of rare genetic blood disorders involving aplastic anaemia associated with a family history.

Familial Hypercholesterolaemia (FH)

5 HaloPlex Caused by mutations in genes for cholesterol metabolism. Premature atherosclerosis and increased risk of CHD.

Osteopetrosis (OP) 21 HaloPlex Diagnosis of very rare disorders characterized by an increased bone density due to impaired bone resorption. Aid decisions on potential curative stem cell transplants.

Steroid Resistant Nephrotic Syndrome (SRNS)

37 HaloPlex A disease of kidney filtration, resulting in massive and unremitting protein loss into the urine. A confirmed genetic diagnosis alters immunosuppressant treatment.

Cancer (BRCA/solid tumour panel)

94 TruSight Genes associated with common (e.g. breast, colorectal) and rare cancers. 284 SNPs found to correlate with cancer predisposition through GWAS.


Current NGS assays• Currently all NGS assays are targeted gene panels looking for point mutations and

gene copy number variation, run on Illumina MiSeq• Agilent SureSelect XT Target Enrichment System

– 3µg input DNA (30ng/µl in 100µl)– 5-6 days wet lab time– Mechanical shearing by sonication using Bioruptor

• Agilent Haloplex– 200ng input DNA (5ng/µl in 45µl)– 3 days wet lab– Fragmentation by enzymatic restriction digestion– Currently 16 indexed libraries pooled

• Agilent TruSight– 50ng input DNA (5ng/µl in 10µl)– 4 days wet lab– Enzymatic DNA fragmentation– 24 indexed samples pooled


http://genoseq.ucla.edu/action/view/File:Miseq-personal-sequencer.png


NGS Considerations• Sample quantity/quality

– DNA extraction method– Accurately quantify input DNA

• Two-step method of DNA normalisation is recommended• Labour intensive protocols; many liquid handling steps

– Confirm presence of product at appropriate intervals– Sample transfer checks– Reagent storage requirements

• Data handling and management– In-house pipeline with bioinformatician support– Mass data storage requirement (Arkivum company pending)

• Post-NGS work– Follow-up primer design; Sanger sequencing to confirm mutations or fill gaps

• Provision for primer storage • A system for location of primers for Sanger confirmations



SureSelectXT

• Generation of libraries from 3µg genomic DNA (relatively high input DNA)– Request 5µg of DNA; at least 50ng/µl

• Allow for variation in DNA concentration between NanoDrop and Qubit (fluorescence signal only when bound to target dsDNA)

• Allow sufficient DNA for confirmatory and follow-up procedures e.g. Sanger sequencing and/or MPLA

• However, ~2µg DNA has worked well (IBMFS, IPN)• Fragmentation of DNA by sonication using a Bioruptor

– Optimum average starting fragment size 150-200bp – Regularly achieved after 60 cycles (30 secs on/30 secs off)– Inadequately sheared gDNA can result in suboptimal data

• Bioruptor LoBind tubes should be checked for abnormalities• Diluted samples should not contain bubbles

– Chemagen-extracted DNA (beads) proved resistant to shearing. • DNA clean-up + re-precipitation solved this problem



SureSelectXT

• 24-hour 65°C hybridisation step– Use a compression pad to minimise evaporation

• Inefficient washing of post-hybridisation libraries can result in a high percentage of off-target reads being maintained within the library preparation and loaded onto the MiSeq

• Residual AMPure beads from SureSelect protocol can give signal which runs with the upper marker – Any signal under the upper marker affects quantification


Troubleshooting HaloPlex


• Mixed results; success, partial fail and completely failed runs

• Highlights the importance of complete QC data• Reagent log• Instrument calibration records• Regular equipment maintenance• Transfer checks• Use of controls

• Agilent supported troubleshooting approach



• DNA samples– Excess EDTA in DNA samples (present in TE buffer for eluting DNA)

can affect the activity of the restriction enzymes• Reagents

– Replace NaOH made up in-house to buy in ready-made– Dilute NaOH using larger volumes to ensure accuracy as the

concentration of 50mM NaOH to elute DNA is critical– Ensure thorough mixing of pooled restriction-digested products to

ensure complete deactivation of restriction enzymes• Carried ECD through whole process• Reduce any possible bead carryover into PCR step

– Changed magnet


TruSight

• New protocol in BGL– TruSight Cancer assay available “off the shelf”, already optimised– Initially BRCA1/BRCA2 only– targets 94 genes suspected to play a role in predisposing to cancer – associated with both common and rare cancers

• Samples pooled earlier in protocol• Increased amounts of formamide • Lower library prep concentrations than expected

– not affected data to date


Breast and Ovarian Cancer

Breast Cancer association

HNPCC – Hereditary non-polyposis colorectal cancer (Bowl Cancer)

Pheochromocytoma - Tumor of the Adrenal gland

Neurofibromatosis - Tumours affecting the nerves

Tuberous sclerosis - Causes mainly benign tumours to grow in different parts of the body


Future Developments

• Move from SureSelectXT to SureSelectQXT protocol– Reduce time;

• No mechanical shearing• Fragment DNA + adapter-tag DNA ends in single enzymatic step

– Less DNA; • Only 50ng input gDNA (3µg with XT)

• Faster throughput– Automation – a tender process for robot in progress– Upscale number of libraries pooled and run on the MiSeq

• Exome sequencing– Clinical exomes; larger panel testing using Nextera



Thank you


Thank you to the BGL Sequencing Teamand

Thank you for listening!

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Documents

exceptional healthcare

ng input dna

sureselect xt genes

sureselect xt diagnosis

dna repair mechanisms

rare disorders

days wet lab fragmentation

primer storage