summer internship project
Post on 20-Sep-2014
698 views
DESCRIPTION
A presentation of my summer internship project at ARUP Laboratories.TRANSCRIPT
![Page 1: Summer Internship Project](https://reader033.vdocument.in/reader033/viewer/2022061104/541c81877bef0a16088b5364/html5/thumbnails/1.jpg)
OPTIMIZATION AND VALIDATION OF THE LIGHTSCANNER® ASSAY FOR THE MCAD
DEFICIENCY DISEASE
Sadhaasivam KamalanaadhanMST Program – Biotechnology Track
u0569466
![Page 2: Summer Internship Project](https://reader033.vdocument.in/reader033/viewer/2022061104/541c81877bef0a16088b5364/html5/thumbnails/2.jpg)
COMPANY BACKGROUND
Founded in 1984.
Enterprise of the University of Utah – Department of Pathology.
Currently employs more than 2600 employees.
Among nation’s leading clinical and pathological reference laboratories.
![Page 3: Summer Internship Project](https://reader033.vdocument.in/reader033/viewer/2022061104/541c81877bef0a16088b5364/html5/thumbnails/3.jpg)
INTERNSHIP OBJECTIVES
Optimize and validate the Lightscanner® Assay for the MCAD deficiency disease.
Prepare an internal database of MCAD mutations.
Learn the process of pricing an assay.
![Page 4: Summer Internship Project](https://reader033.vdocument.in/reader033/viewer/2022061104/541c81877bef0a16088b5364/html5/thumbnails/4.jpg)
PROJECT BACKGROUND MCAD – Medium-chain acyl-CoA dehydrogenase deficiency
(ACADM gene) Autosomal Recessive hereditary disease. Most common mutation
being c.985A>G.
Impairs beta-oxidation of medium-chain fatty acids.
1/15,000 live births in the USA.
Symptoms include lethargy, vomiting, hypoglycemic coma.
Treatable through diet management.
Fatal if not diagnosed early.
![Page 5: Summer Internship Project](https://reader033.vdocument.in/reader033/viewer/2022061104/541c81877bef0a16088b5364/html5/thumbnails/5.jpg)
![Page 6: Summer Internship Project](https://reader033.vdocument.in/reader033/viewer/2022061104/541c81877bef0a16088b5364/html5/thumbnails/6.jpg)
ASSAY WORKFLOW
![Page 7: Summer Internship Project](https://reader033.vdocument.in/reader033/viewer/2022061104/541c81877bef0a16088b5364/html5/thumbnails/7.jpg)
TECHNICAL BACKGROUND Scanning assay is based
on Hi-Res Melting® for mutation scanning. DNA is denatured or
‘melted’. Special dye whose
fluorescence decreases as DNA becomes single stranded – real time melt curves
Lightscanner® detects differences between melt curves of different DNA.
Source: http://www.path.utah.edu/news/hi-res-dna-melting-analysis/image/image_view_fullscreen
![Page 8: Summer Internship Project](https://reader033.vdocument.in/reader033/viewer/2022061104/541c81877bef0a16088b5364/html5/thumbnails/8.jpg)
Source: http://en.wikipedia.org/wiki/File:STD_HRM_plot.JPG
![Page 9: Summer Internship Project](https://reader033.vdocument.in/reader033/viewer/2022061104/541c81877bef0a16088b5364/html5/thumbnails/9.jpg)
OPTIMIZATION OF ASSAY – PCR CONDITIONS ACADM gene has 12 exons.
Exon specific primers designed by Idaho Technology Inc.
Amplify exons with a mix of Lightscanner® Master Mix, primers and mineral oil overlay.
Thermocycling conditions were optimized using a touchdown PCR protocol on the BioRad iCycler® (Ramp rate 3.3°C/second)
![Page 10: Summer Internship Project](https://reader033.vdocument.in/reader033/viewer/2022061104/541c81877bef0a16088b5364/html5/thumbnails/10.jpg)
AGAROSE GEL ELECTROPHORESIS
![Page 11: Summer Internship Project](https://reader033.vdocument.in/reader033/viewer/2022061104/541c81877bef0a16088b5364/html5/thumbnails/11.jpg)
OPTIMIZATION OF ASSAY - LIGHTSCANNER®
Melting temperature range set between 70°C – 95°C.
Normalized melt curves to yield tight fitting of curves.
DNA extraction methods affect the melt curve analysis.
Lack of mineral oil, presence of bubbles etc. affect performance.
![Page 12: Summer Internship Project](https://reader033.vdocument.in/reader033/viewer/2022061104/541c81877bef0a16088b5364/html5/thumbnails/12.jpg)
![Page 13: Summer Internship Project](https://reader033.vdocument.in/reader033/viewer/2022061104/541c81877bef0a16088b5364/html5/thumbnails/13.jpg)
VALIDATION - PRECISION
Defined as the reproducibility between replicate measurements. Within Runs – How well does instrument perform when
same sample is tested repeatedly? Between Runs – How well does instrument perform when
same samples are tested between different days, instrument, operating conditions etc.?
CM#1 – Wild type ACC#1 – Patient sample Day1: CM#1 vs. ACC#1 Day2: CM#1 vs. ACC #1 Day3: CM#1 vs. ACC#1, ACC#1, ACC#1
(triplicates of Acc#1 vs. CM#1)Within
Between
![Page 14: Summer Internship Project](https://reader033.vdocument.in/reader033/viewer/2022061104/541c81877bef0a16088b5364/html5/thumbnails/14.jpg)
Exons 4 & 5 – within runs
![Page 15: Summer Internship Project](https://reader033.vdocument.in/reader033/viewer/2022061104/541c81877bef0a16088b5364/html5/thumbnails/15.jpg)
Problematic exons 8 &12 – Within Runs
![Page 16: Summer Internship Project](https://reader033.vdocument.in/reader033/viewer/2022061104/541c81877bef0a16088b5364/html5/thumbnails/16.jpg)
Instrument Calls
Operator Calls
n – negative, p – positive, ?n – questionable negative
![Page 17: Summer Internship Project](https://reader033.vdocument.in/reader033/viewer/2022061104/541c81877bef0a16088b5364/html5/thumbnails/17.jpg)
VALIDATION - ACCURACY
Concordance of results using two different assays. Analytical Sensitivity –
How sensitive is the assay? True Positives/(True Positives+ False Negatives)
Analytical Specificity – How specific is the assay? True Negatives/(True Negatives + False positives)
![Page 18: Summer Internship Project](https://reader033.vdocument.in/reader033/viewer/2022061104/541c81877bef0a16088b5364/html5/thumbnails/18.jpg)
SETUP OF ACCURACY RUNS
25 de-identified samples KS#1 to KS#25 run over 4 days vs. Wild-type CM#1 Day4: CM#1 vs. KS#1 to KS#6 Day5: CM#1 vs. KS#7 to KS#12 Day6: CM#1 vs. KS#13 to KS#18 Day7: CM#1 vs. KS#19 to KS#25
Operator vs. Instrument Calls
Compared with existing sequencing data.
![Page 19: Summer Internship Project](https://reader033.vdocument.in/reader033/viewer/2022061104/541c81877bef0a16088b5364/html5/thumbnails/19.jpg)
![Page 20: Summer Internship Project](https://reader033.vdocument.in/reader033/viewer/2022061104/541c81877bef0a16088b5364/html5/thumbnails/20.jpg)
OPERATOR ASSISTANCE REQUIRED
![Page 21: Summer Internship Project](https://reader033.vdocument.in/reader033/viewer/2022061104/541c81877bef0a16088b5364/html5/thumbnails/21.jpg)
![Page 22: Summer Internship Project](https://reader033.vdocument.in/reader033/viewer/2022061104/541c81877bef0a16088b5364/html5/thumbnails/22.jpg)
Instrument Scores
Operator Scores
![Page 23: Summer Internship Project](https://reader033.vdocument.in/reader033/viewer/2022061104/541c81877bef0a16088b5364/html5/thumbnails/23.jpg)
OVERALL – INSTRUMENT VS. OPERATOR
Instrument Operator
![Page 24: Summer Internship Project](https://reader033.vdocument.in/reader033/viewer/2022061104/541c81877bef0a16088b5364/html5/thumbnails/24.jpg)
Exon Results
![Page 25: Summer Internship Project](https://reader033.vdocument.in/reader033/viewer/2022061104/541c81877bef0a16088b5364/html5/thumbnails/25.jpg)
![Page 26: Summer Internship Project](https://reader033.vdocument.in/reader033/viewer/2022061104/541c81877bef0a16088b5364/html5/thumbnails/26.jpg)
![Page 27: Summer Internship Project](https://reader033.vdocument.in/reader033/viewer/2022061104/541c81877bef0a16088b5364/html5/thumbnails/27.jpg)
Can I ever become
independent?
Someday maybe you
will!
INSTRUMENT (DEJECTED)
OPERATOR(REASSURING)
![Page 28: Summer Internship Project](https://reader033.vdocument.in/reader033/viewer/2022061104/541c81877bef0a16088b5364/html5/thumbnails/28.jpg)
VALIDATION SUMMARY FOR EXONS
![Page 29: Summer Internship Project](https://reader033.vdocument.in/reader033/viewer/2022061104/541c81877bef0a16088b5364/html5/thumbnails/29.jpg)
DISCUSSION AND RECOMMENDATIONS
Operator judgment is needed to increase sensitivity of the assay. Problematic when there are large amount of samples to be
analyzed
Further optimization for the assay is needed. Software settings optimization Redesigning primers Consistency in DNA extraction methods
Assay can be used along with sequencing.
![Page 30: Summer Internship Project](https://reader033.vdocument.in/reader033/viewer/2022061104/541c81877bef0a16088b5364/html5/thumbnails/30.jpg)
INTERNSHIP OBJECTIVES
Optimize and validate the Lightscanner® Assay for the MCAD deficiency disease.
Prepare an internal database of MCAD mutations.
Learn the process of pricing an assay.
![Page 31: Summer Internship Project](https://reader033.vdocument.in/reader033/viewer/2022061104/541c81877bef0a16088b5364/html5/thumbnails/31.jpg)
MUTATION DATABASE
Why the need for an internal database? Huge amount of literature sources, databases for mutations
available. Condensing the data helps the clinical lab.
About the database. Created in MS-Excel after streamlining mutations from
various sources such as HGMD, NCBI etc. Contains MCAD gene sequence, nucleotide and codon
position of mutations and links to publications /references. Mutations categorized into Missense/Nonsense, Splice
mutations, deletions and insertions.
![Page 32: Summer Internship Project](https://reader033.vdocument.in/reader033/viewer/2022061104/541c81877bef0a16088b5364/html5/thumbnails/32.jpg)
![Page 33: Summer Internship Project](https://reader033.vdocument.in/reader033/viewer/2022061104/541c81877bef0a16088b5364/html5/thumbnails/33.jpg)
INTERNSHIP OBJECTIVES
Optimize and validate the Lightscanner® Assay for the MCAD deficiency disease.
Prepare an internal database of MCAD mutations.
Learn the process of pricing an assay.
![Page 34: Summer Internship Project](https://reader033.vdocument.in/reader033/viewer/2022061104/541c81877bef0a16088b5364/html5/thumbnails/34.jpg)
PRICING COMPONENTS
![Page 35: Summer Internship Project](https://reader033.vdocument.in/reader033/viewer/2022061104/541c81877bef0a16088b5364/html5/thumbnails/35.jpg)
COST ACCOUNTING
Documents the actual cost of performing the test.
Helps maintain inventory.
Two types of costs involved. Direct Costs - Cost of reagents, supplies, man-hours, royalties
Factored into a pre-made spreadsheet.
Indirect Costs - Costs associated with running the lab – Refrigeration, Electricity, General maintenance etc. Determined by the accounting department.
![Page 36: Summer Internship Project](https://reader033.vdocument.in/reader033/viewer/2022061104/541c81877bef0a16088b5364/html5/thumbnails/36.jpg)
COMPETITOR ANALYSIS
Lets us know what competition offers for the same test in terms of price, features etc.
Helps ARUP to be competitive. ARUP hotline – R&D to Clinical Lab.
Spreadsheet containing following details Background of disease and the test. Name of Lab/Company performing the test. Internal test code and CPT code. Name of the test and the methodology used for testing. Price of the test offered by competition plus comments.
![Page 37: Summer Internship Project](https://reader033.vdocument.in/reader033/viewer/2022061104/541c81877bef0a16088b5364/html5/thumbnails/37.jpg)
SAMPLE COMPETITOR ANALYSIS REPORT
![Page 38: Summer Internship Project](https://reader033.vdocument.in/reader033/viewer/2022061104/541c81877bef0a16088b5364/html5/thumbnails/38.jpg)
(Black = Input; Blue = Formulas)
Description Driver $
Direct & Indirect Testing Costs per BU 50.00 Variable Overhead Costs 15.0% 7.50 Fixed Overhead Costs 0.0% - Net Margin Markup Factor 12.0% 6.00
Minimum Price, Excluding Add-On's 27.0% 63.50
Add-On's:Logistics 3.00 Royalties 66.50 16.63 20.0%
Minimum Price, Including Add-On's 83.13
If test is subject to a royalty, insert royalty % here.
PRICING SPREADSHEET - EXAMPLE
![Page 39: Summer Internship Project](https://reader033.vdocument.in/reader033/viewer/2022061104/541c81877bef0a16088b5364/html5/thumbnails/39.jpg)
CLIENT PRICING
![Page 40: Summer Internship Project](https://reader033.vdocument.in/reader033/viewer/2022061104/541c81877bef0a16088b5364/html5/thumbnails/40.jpg)
CONCLUSION – THE STORY OF AN ASSAY
![Page 41: Summer Internship Project](https://reader033.vdocument.in/reader033/viewer/2022061104/541c81877bef0a16088b5364/html5/thumbnails/41.jpg)
THANK YOU