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Mónica Monte

HID FAS Spain&Portugal

The Nuts and Bolts behind our Solutions to your Latest Laboratory Challenges

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Agenda

• 3500 Data Collection 4.0

o Signal Optimizationo Pullup Reduction

o Off-scale Data Recoveryo Additional Features

• GMIDX 1.6

o Off-scale Data Recoveryo Export for Probabilistic Genotyping

o Sorting with the Softwareo Command Line Interface

• Converge 2.1 o Mito DNA Analysis Module

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The Exterior: Data Collection v4.0 and GeneMapper IDX v1.6

Improve Data Interpretation

• Reduce pull-up edits

• Improve 1st pass success

• Signal Optimization

• More efficient data transfer

to probabilistic genotyping

Improve the User Experience

• Added flexibility for the

3500 series instruments

• Streamlined workflow

• Support languages

• 6-dye installation standard

• Feature requests

IT Compliance

• Win 10 support

• No licensing

• CODIS compatibility

• Internationalization

Decrease Decrease Analysis

Time

Improve Efficiency

Support Support Flexible

Workflows


Signal optimization

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Signal Optimization

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Signal Optimization

• Goal: reduce capillary-to-capillary variation within an injection

• Dual-focused approach developed

Signal optimization

• Utilize spatial calibration data

• 24-capillary instruments only

• Automatically applied

1. Optical approach: Reduce variation introduced by optics

• Optimize injection conditions in the run module

• Both 8- and 24-capillary instruments

• Optional setting

2. Injection approach: Reduce variation introduced by injection conditions

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• Displayed in Spatial Calibration screen

• Included in the Spatial calibration report, can be exported or printed

• 8 capillary instruments: factor = 1 for all capillaries

Signal Optimization Factor

Optical Approach: Reduce Variation Due to Optics

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Injection Approach: Reduce Variation Due to Injection

• Choose an assay: _SO suffix

• Contains instrument protocol: _SO suffix

• Contains run module: HID36_POP4(xL)_SO

New signal optimization (SO) run module

• Injection conditions similar to sequencing applications

• Position of capillary in sample optimized

• A small amount of POP is introduced into the sample prior to injections

• These conditions have been demonstrated to improve uniformity across capillaries

Enhanced run module

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Signal Optimization Combined

Combination of both methods shows greatest decrease in variation for 24 capillary instruments


Pull-up Reduction

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Recognizing Pull-up

• Analyzed data: centered under parent peak

• Raw data: same shape as parent peak

Spectral

• Analyzed data: not centered under parent peak

• Raw data: sinusoidal curve

Instrument-Specific

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Pull-Up Reduction in DC v4.0

Pull-up reduction

Addresses spectral (including off-scale)

and instrument-specific pull-up

Utilizes sample-specific spectral data

To enable pull-up reduction

Utilize an Applied Biosystems (AB) STR

kit

Perform spectral calibration using an

AB dye matrix and AB dye set

Choose applicable AB assay

Spectral correction

Takes place prior to creation of .hid files

Raw Data viewed in GeneMapper IDX (all versions) will have the

reduction applied

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Pull-Up Reduction in DC v4.0

• Identified by labeling as allelic ladder sample type

• Identified by density of peaks in profile

Allelic ladders – pull-up reduction DISABLED

• Signal-to-noise ratio

• Number of peaks and dyes detected

• Calibrated matrix will still be applied (ie sample-specific spectral data not utilized due to poor quality)

Poor quality samples – pull-up reduction DISABLED

• Adds approximately 2-3 minutes per injection for processing (3500xL)

• Pull-up frequency is reduced

• Peak height percentage of remaining pull-up generally falls within 1-3%

Important Notes

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Improved Data Interpretation – Pull-up

Single-source 1ng sample amplified with the GlobalFiler kit analyzed at 50 RFU

31 pull-up edits

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Improved Data Interpretation – Pull-up Reduction

The same GlobalFiler profile analyzed at 50 RFU after pull-up reduction is applied.

6 pull-up edits


Off-scale data recovery

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• Capture “off-scale”

• Truer peak height

• Decrease manual interpretation

• Off-scale data now approaching

~65,000 RFU

• Stutter filters more correct

• When using Global filters these are

more effective for reducing unspecific background signals

• Decrease pull-up associated with off-scale peak

• On-scale data not impacted

• Higher 1st pass success rate

Off-Scale Data Recovery (OSR) for Database Samples

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Off-Scale Data Recovery (OSR)

• Bin data signals saturation

• Bin data converted to dye data

• Dye data capped at ~32K RFU values

Saturation

• CCD camera saturation limit is extended

• More bin data can be utilized

• Dye data capped at ~65K RFU values

Off-Scale Data Recovery

GMIDX 1.6 is required to analyze/view OSR data above ~32K RFU.

OSR Instrument Protocol

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65K

32KNo OSR

OSR

GMIDXv1.6 raw data collecting without and with OSR (reinjection of same sample)

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3500 Data Collection v4.0: Off-Scale Recovery

IDX v1.6 Database sample, GFE

35K

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3500 Data Collection v4.0: Off-Scale Recovery

BEFORE: 10 Off-scale markers, 16 Marker level flags

IDX v1.6

45K

IDX v1.6

NOW: 0 Off-scale markers, 0 Marker level flags

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Under the Hood: Off-Scale Data Recovery (OSR)

• Robust, single source profiles

• High thresholds

• Global filter

Developed for database samples

• Cross talk

• Carryover

• PCR-related artifacts

• Peak height plateau due to reagent consumption

Increased DNA input causes

Not intended for casework samples

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Off-Scale Data Recovery Field Testing Results

OSR Disabled(Any6Dye dye set)

OSR Enabled(J6 dye set)

Test Site 1 5 markers in 2 samples 0 markers in 0 samples

Test Site 3 99 markers in 12 samples 4 markers in 1 sample

Test Site 2 5 markers in 2 samples 0 markers in 0 samples

(10) Reference GFE samples(3) GFE Positive Controls

Data from Test Site 1

OSR ON

OSR OFF

Using OSR dye set enables OSR feature – works with AB dyes


Additional Features

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Flexible Plate Loading - 3500 Series

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Added Flexibility and Control – System Preferences

• Instrument settings

• Uncheck “allow runs” options

• Access: admins only

• Results in pre-run validation check error

•Choose: hard stop

Admin view

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DC 4.0 Enhancements - Summary

Improved data analysis options

• Pull-Up Reduction

• Off-Scale Data Recovery (OSR)

• Signal Optimization (SO)

Improved user experience

• No yearly licensing

• Flexible Plate Loading

• 6-dye HID Install Standard

• Library Assets Updated

• Flexible admin preferences for hard stops and warnings

• Bi-weekly scheduling

• Exportable consumables list

• Exportable injection list

• Normalization Factor and average peak height in Sample View

• EPT for completed/aborted runs

User Bulletin available


GeneMapperTM ID-X Software v1.6

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In the Driver’s Seat with GMIDX v1.6

Windows 10

64-bit Compatibility

Windows 7

64-bit

Compatibility


Data Export for Probabilistic Genotyping

Software

Latest CODIS support

Command Line Interface (CLI)

Updates

Enhanced User Interfaces

Printing Enhancements

Sorting Improvements

SeqStudio File Support

Profile Comparison Tool

Updates

Support for Chinese

Language

HID validation expected end of 2019

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• Upper limit: ~65,000 RFU

• MUST use GeneMapper™ IDX v 1.6

RFU upper limit extended for DC 4.0

• Global filters more effective

• Stutter filters more applicable

• Decreased pull up

• Higher 1st pass success rate

• On-scale data not impacted

Better peak height estimate

• Peaks will be capped

• Secondary dye colors may be pulled-up to 65K

• Analyze with caution!

Severely off-scale data (>65K RFU)

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41251

29620

v1.6 = Pass

v1.5 =

Flagged

OS w/ pink

bar


Export for Probabilistic Genotyping

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Probabilistic Genotyping – Data Export

• Two export file type options:• .txt or .csv

• Select from the right-hand drop-down menu

• When you export without stutter:• Labeled allele peaks are identified

as Allele 1 and Allele 2

• When you export with stutter:• Stutter peaks and labeled allele

peaks are labeled sequentially


Enhanced Plot and Printing Options

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Printing

Custom Header

Marker header outlined in color

Bin Color adjusted to improve visibility


Sorting within the Software

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• Projects sorted by default with most recently created on top

• Analysis Methods and Size Standard drop downs in project window are alphabetical

• Genotype Table can be sorted by Marker only without dye consideration

General Sorting

Sorting Within the Software

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Comparison NGM Detect - NGMSelect

Different Samples - One Marker – Different dyes

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Comparison NGM Detect - NGMSelect

Different Samples - One Marker - sample order by selection


Command Line Interface

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Commend Line Interface (CLI) Updates

Export/ Import database

Process multiple commands with single login and single command

Export sample information for a project or individual sample

Create a project from samples in different projects

Printing: Page orientation, paper size, pdf file name

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GMIDX v1.6 Details- Summary

Improved data analysis options

• Off-Scale Data Recovery (OSR)

• Data export for stutter

• Peak height detection enhancement

Improved user experience

• Profile Comparison Tool

• CLI

• Sorting (projects, drop-down menus, Genotypes Table)

• Plots (scrolling, editing/deleting, size standard display, PLI, tables)

• Panel manager (v6x, IQS and Ymarker functionality)

• Report manager

• SS Normalization

• Printing (comment, marker header)

• SAE: Audit record deletion, log-in list

• Seq Studio file support

IT compliance

• Win 7 or Win 10

• CODIS updates

• Chinese Language

• Oracle Security

User Bulletin available


The Applied Biosystems mtDNA NGS workflow

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When current CE-based methods can’t resolve a case…

…NGS can supplement traditional CE STR testing with

forensically relevant, targeted panels for specific uses.

• Low quality and low quantity samples (mtDNA)

• Precision ID mtDNA Whole Genome Panel

• Precision ID mtDNA Control Region Panel

• Degraded samples (identity SNPs)

• Precision ID Identity Panel

• Investigative leads (ancestry and phenotypic SNPs)

• Precision ID Ancestry Panel

• Ion AmpliSeq™ DNA Phenotyping Panel

• Complex Mixtures (NGS STRs)

• Precision ID GlobalFiler™ NGS STR Panel v2

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Simplest targeted NGS workflow

• Total hands-on time: less than 45 minutes

• Total overall time: as little as 25 hours

• Converge Software analysis modules for mtDNA, SNPs and STRs

• HPS validation services now available for Precision ID NGS System

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Converge Now Provides Unified NGS Analysis Solution

35 CODIS Core +

Expanded Loci

v2

Version 2.1

w/ Ion GeneStudioSeries & Legacy

S5/S5xl

HW/SWSupport

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• Algorithm that utilizes haplogroup information and context

sensitive information for enhanced alignments

• Nucleotide numbering to circular version of revised Cambridge Reference Sequence (rCRS) mtGenome

• Comparison to rCRS to support haplotype designations using

standard forensic nomenclature (ISFG and IUPAC recommendations)

• Highly accurate variant detection w/ coverage plots

• Assessment of amplicon coverage, variant classifications

• Link to Mito IGV for detailed variant analysis and export w/ different file formats (.xls, FASTA)

• Haplotype profile comparisons within a batch and case

• Identification of mtDNA heteroplasmy and NUMTs

• Haplotype and Haplogroup reporting with EMPOP interface for profile searches (to support forensic casework & phylogenetic

analysis)

mito features

Mito Analysis on Converge v2.1

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Thank you for your time & attention

© 2018 Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified. Windows is a trademark of

Microsoft Corporation.

Products described herein are intended for forensic or paternity use only. Not for use in diagnostics procedures.

The content provided herein may relate to products that have not been officially released and is subject to change without notice.

The Nuts and Bolts behind our Solutions to your Latest ... › content › dam › LifeTech ›...

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