Exeter s experience of trying to keep up with demand for NGS. Anna Bussell
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1 Exeter s experience of trying to keep up with demand for NGS Anna Bussell
2 Overview Sample received and DNA extracted Admin & test review NGS wet lab work Sequencing Report authorisation & distribution Data import & report generation Data analysis Bioinformatic data processing
3 Services provided Targeted NGS Exome sequencing NEXTFlex DNA rapid seq kit Pre hybe indexing able to pool samples o 6 x DIAG o 12 x Pancreatic Sub virtual panels to prevent IFs <10 genes = 56 calendar days >10 genes = 112 calendar days Gene agnostic approach to diagnose rare monogenic severe prenatal or paediatric onset disorders Agilent SurselectXT Target Enrichment Trio/couple/singleton bioinformatic pipelines P 112 days TAT, aim for 2-3 weeks
4 Wetlab process tngs Day 1: Qubit QC (2hrs) Fragmentation & QC (3.5hrs) Day 2: End repair + A-Tail (1hr) Adaptor ligation & Prehybe Indexing (2hrs) PCR & QC (2hrs) Day 3: Sample pooling and drying down (3hrs) Hybridisation set up (1.5hrs) *Hybridisation reaction happens overnight* Day 4: Capture & wash (4hrs) PCR (2hrs) Qubit & TS2200 QC (1hr) Day 5: Project pooling (1 hr) Nextseq500 set up (1 hr) Exome Day 2: A-Tailing, Ampure (1.5hr) Day 1: Qubit QC (1 hr) Fragmentation, ampure & QC (3hrs) End repair, AMPure (2.5hrs) Adaptor ligation, Ampure (1.5hr) PCR, Ampure (1.5hr) TS2200 QC (0.5hr) Pooling & drying (1hr) Hybridisation set up (1hr) Day 3: Capture & wash (4hrs) PCR and indexing, AMPure (2 hrs) Qubit QC (1hr) Day 4: TS2200 QC (2hrs) Project pooling (1 hr) Nextseq500 set up (1 hr) *Hybridisation reaction happens overnight*
5 # of samples received They just keep coming Samples for exome sequence analysis Month tngs exome patients (407 families), diagnostic yield of 42%
6 Where have we improved? Sample received and DNA extracted Admin & test review NGS wet lab work Sequencing Report authorisation & distribution Data import & report generation Data analysis Bioinformatic data processing
7 Illumina Hiseq2500 Located on the Streatham Campus Heavily relied on Exeter sequencing service High output run = 12 days Illumina Nextseq500 Located in the RILD building Nearly full control High output run takes 29hrs >100 runs so far
8 Where have we improved? Sample received and DNA extracted Admin & test review NGS wet lab work Sequencing Report authorisation & distribution Data import & report generation Data analysis Bioinformatic data processing
9 Sample throughput More clinicians are opting for NGS testing tngs barcodes increased from 96 to 288 Exome barcodes increased from 16 to 96 Formulas in place to highlight clashes between the two
10 Automation 1 sample at a time to 88 samples in 3 mins! In progress due to liquid detection issues. + Upgraded the Agilent Bravo = walk away for 3 hours! Additional Eppendorf robot = 48 samples at a time. Able to run tngs and exomes in parallel
11 Employment & training More staff = resilience
12 Where can we improve? Sample received and DNA extracted Admin & test review NGS wet lab work Sequencing Report authorisation & distribution Data import & report generation Data analysis Bioinformatic data processing Stopped sanger confirmations for nonsense & missense variants Data import to become more automated. Database that can handle sample management rather than excel sheets
13 % witin TAT Number of repotrs Turn around times 100% % 80% % 60% % % 30% % 50 # of reports issued 10% 0% Jan Feb Mar Apr May Jun Jul Aug Sept Oct % of NGS reports within TAT 0 % of reported within TAT
14 Summary Exeter provides a variety of NGS services New equipment and more staff has improved TATs and resilience Room for further improvement: introduce more automation and integrate LIMS.
15 Acknowledgements Dr Richard Caswell Dr Matthew Johnson Andy Parrish Dr Lucy Malin Dr Thomas Laver Dr Matthew Wakeling Jessica Settle Lewis Pang Helen Webb Sian Ellard Diagnostic Molecular Genetics team Exeter University Medical School Exeter Sequencing Service
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