Massively Parallel Sequencing - integrating the Ion PGM™ sequencer into your forensic laboratory

Massively Parallel Sequencing -integrating the Ion PGM™

sequencer into your forensic laboratory.

| Dr SallyAnn Harbison

March 2015

Its not all about the science- its also about the people.

What is it that we are replacing?

National laboratory with Research and Development function, the sole provider to NZ police

National DNA Profile Databank

4 STR Multiplexes, Globalfiler® kits, SGMPlus® kits , PowerplexY ® kits, Minifiler™ kits

Continuous models for DNA interpretation

34 cycle SGM Plus® kit Low Copy Number testing

mRNA profiling for body fluid identification

Laser Micro-dissection including XY FISH.

Where are we now?I think we are all agreed that:

Our existing platforms are very sensitive

They are well characterised

We know how they work and what to try when they don ’t

We have methods to interpret the data

We’re all (more or less) doing the same thing

Turn around times for samples and cases can be quic k (1-2 days)

Evidence is (generally) accepted in court.

What are we looking for?

A robust well characterised technology, supported b y the forensic community

That is compatible with the NZ DNA Profile Databank

That approaches the sensitivity of existing STRs

That delivers more information per sample

Bio-informatic pipelines to interpret the data

With a reasonably fast sample through put time less than 5 days, automation

That is cost neutral.

Our project:

A project with three objectives:

�Beginning the transformation of (routine) STR profiling to genomic technologies

�Adding additional SNP markers (e.g. ancestry informative, hair, skin and eye colour, mtDNA, serology) in a single workflow

�Non Human DNA testing- Species identification from difficult and mixed samples expanding our range of services.

Evaluate MPS technology’s ability to sequence forensic markers� Is it possible to sequence samples already

amplified with Identifiler® and PPY® kits?� Design and test a custom panel of markers

including most of the existing forensic markers.

Sequencing Identifiler ® + PPY® products

Amplify STRs with Identifiler® and PPY® kits[5x duplicates plus two mixtures]

Extract and quantify DNA from samples

Amplicons purified and quantified[Agencourt® AMPure XP® and Agilent 2100 Bioanalyzer]

Ion PGM™ workflow

Ion PGM™ WorkflowAdd Ion PGM™ adaptors and barcodes [KAPA© Ion Torrent DNA library prep kit]

Quantify (Ion Library Quantification kit) and pool libraries (26pM)

Template Preparation and enrichment

[Ion PGM™ OT2 400bp kit]

Sequence on Ion PGM™ 316 chip

Amplicons purified and checked[Agencourt® AMPure® XP and Agilent 2100 Bioanalyzer]

Bioinformatic WorkflowInitial QC was carried out on the Torrent Server

Quality assessment using FastQC and Solexa QA

Alignment programs Bowtie2 and STRait razor v1.2

FastQ quality trimmerPhred score of 25 and minimum length of 40bases

Sequence quality and alignment

Whilst 76% of the reads aligned with the STR sequences using Bowtie 2, only 1/3rd of these spanned the entire repeat region and were able to be aligned with STRait Razor.

Improving the quality/ length of the sequence reads to maximise allele calling is a key area of development

Sample 1a. FastQC quality score across all nucleotides of sequencing reads.

Establishing some starting guidelines for calling alleles

In each sample

> 50 reads must be aligned to a given locus (minimum coverage threshold)

>10 reads or a defined % of the total reads for the locus equivalent to 10 reads was required for an allele to be called

CE based stutter values

Heterozygote locus balance of 50%.

Calling Genotypes- single source samples

Some Observations

FGA Reads Call Length Sequence

19.1 24

19.2 89

19.3 1250 FGA:19.3 79 bases

TTTCTTTCTTTCTTTTTCTCTTTCTTTCTTTCTTTCTTTCTTTCT

TTCTTTCTTTCTTTCTTTCTTTCTCCTTCCTTCC

20 1189 FGA:20 80 bases

TTTCTTTCTTTCTTTTTTCTCTTTCTTTCTTTCTTTCTTTCTTTC

TTTCTTTCTTTCTTTCTTTCTTTCTCCTTCCTTCC

In all 7 novel sequence variations found:• Four at D2S1338• 1 each at D8S1179, D3S1358, D19S433 and

D21S11

and 35 examples of allelic discrimination in our 5 people.

Uneven representation of reads across loci compared to the

electropherograms

female/

female

mixture

2:1

Allele Number of

reads

aligned

predicted

major

genotype -

MPS

predicted

minor

genotype -

MPS

RFU major

genotype

predicted

from RFU

minor

genotype

predicted

from RFU

known

major

genotype

known

minor

genotype

FGA 19.3/20 184 0,0 0,0 720 22,27 20,20 22,27 20,20

FGA 21.3/22 227 925

FGA 26.3/27 17not

detected923

total574

DNA multiplexed in one Ion Ampliseq Library preparation for Ion PGM™sequencing from each of 8 people.

Our panel amplifies 280 targets in a single reaction • Amplicons range from 100-300bp in length

• 67 STRs – including all the autosomal and Y STRs contained in the commercial kits, some X STRs and rapidly mutating Y STRs.

• 211 SNPs including: Individual identification SNPs, AIMs, Phenotype (hair, eye, skin), ABO.

• And amelogenin

A custom panel of markers including many existing f orensic markers

Primer Design and Sample Preparation

STR amplicons <= to 275bpSNP amplicons> 100base pairs

Primer panel design for candidate STRs and SNPs using the Ampliseq™ Designer software

https://amplicon.com

DNA from 8 unrelated donors processed in duplicate

Ion PGM™ workflow

Ion PGM™ WorkflowAmplify targets add adaptors and barcodes –

Ion Ampliseq™ Library Preparation Kit

Quantify (Ion Library Quantification kit) and libraries pooled (100pM)

Template Preparation and enrichment [Ion PGM™ OT2 400bp kit]

Sequence on Ion PGM™ 318 v2 chip

Amplicons purified [AMPure XP x2]

Quality Check [2100 Bioanalyzer]

Bioinformatic Workflow

Adaptor removal, barcode sorting and initial QC Torrent Server

Quality assessment using FastQC and Solexa QA

Alignment programs Bowtie2 and STRait razor v1.2

FastQ quality trimmerPhred score of 25 and minimum length of 40bases

Sequence quality and alignment

76% of the reads aligned with either an STR or SNP sequence using Bowtie 2

But only a small proportion (10%) of these spanned an STR repeat region and were able to be aligned with STRaitRazor.

Whereas 80% of these aligned with the SNP sequences

On average 92%

of SNPs were

detected, ranging

from most of

them (206) to

some of them

(121).

In amplicon order in the

custom primer panel

CPP In amplicon order in

the Identifiler multiplex

In amplicon order in the

Identifiler multiplex

� DYS438 + 5 SNPs were not detected in any sample

� DYS392 and Penta E amplified off the repeat target. The detected sequence aligns close to the repeat but does not include the repeat

� DYS389 I, DYS389 II and DYS448 – the sequenced amplicon ends in flanking region and could not be recognized by STRait Razor

� DYS385a, DYS385b and DYS458 – the sequenced amplicon did not contain the full repeat region

Some more details

Quality management, guidelines and standards

Personnel- qualifications, training and competence

Accommodation and environmental conditions

Selection and validation of methods

Control of data

Quality Control procedures

Interpretation and reporting

Guidelines and Standards

Guidelines for laboratory accreditation of MPS (NGS) NATA Technical Note 37 updated October 2014

Ethical and legal issues

Wet lab matters

Bioinformatics

Reporting

Infrastructure

These guidelines can be accessed from the following links:

http://www.nata.com.au

and

http://www.rcpa.edu.au/Library/College-Policies/Guidelines/Implementation-of-

Massively-Parallel-Sequencing





Control of data



People and relationships we will need:Different skill sets are needed- re-training and/or recruitment of scientists

Access to, or employment of, a Bioinformatician or 2

Access to, or employment of, a Statistician or 2

Sequencing specialists to run the sequencers and maintain currency with technology

Good relationships with suppliers

Training and awareness programmes for:

Managers, customers, the public?





Control of data



Lab design - work areas

DNA Extraction and Quantitation

Amplicon PCR

Library Preparation

Template Preparation

Sequence

Analyse

Emulsion PCR step

and particle recovery

Sequencing

Pre PCR

PCR of targets

Library PCR

Bioinformatics

MPS

Pre PCR

Multiplex amplification

Capillary Electrophoresis

Profile Analysis

CE





Control of data



Selection of methods

Performance of the sequencing chemistry- a balance between many factors

A commercially provided kit of DNA markers (eg HID Ion AmpliSeq™ Identity Panel ) or a custom-designed primer set

Select a subset of these DNA markers (eg HID-Ion Ampliseq™ Ancestry Panel ) or go for broke on every sample

DNA Extraction and Quantitation

Library Preparation

Template Preparation

Sequence

Analyse

Steps which are likely to introduce bias

PCR of amplicons

Barcoding/ adapting

Ligating adapters to PCR products, unless already included at the 5’ end of your PCR primers

Library amplification- if used

5-10 PCR cycles of adapted and barcoded amplicons

Ion Library Quantitation Kit (qPCR)

Amplification of the fragments on each ISP bead





Control of data



The first problem in sequencing data analysis

Can / should you keep the raw data from the instrument?

If so, how long for and how?

Do you keep the data after reads of less quality than your QC settings are trimmed?


Do you keep the aligned data only and discard the rest?


Do you keep only the allele calls and sequence?

What about incidental findings?

X millionsTarget seqAdaptor seq Adaptor seqB/C seq

Lots of different options

On platform

for example –the Torrent Server

Open source software command line driven programs, such as:

SAMtools, Burrows-Wheeler Aligner (BWA), and Genome Analysis Tool Kit (GATK), Integrative Genomics Viewer (IGV), Bowtie , Galaxy and GenomeMapper

STR alignment

STRait Razor : [Warshauer et al (FSI: Gen 7 (2013) 409–417).]

Torrent Suite v4.0.2 with the HID_STR_Genotyper (v2.0), FastQC (v3.4.1.1) and

coverage Analysis (v4.0-r77897) plugins (Thermo Fisher).. [S.L. Fordyce et al. /

FSI: Gen 14 (2015) 132–140]





Control of data



Controls you might consider

Extraction

Amplicon PCR

Library PCR

Template PCR

Sequencing

For each stage a positive and negative control?

= 6-8 controls

= 6-8 barcodes used

Extraction +ve and –ve

Amplicon/Library +ve and –ve

Template +ve and –ve

Appropriate (positive ) controls might be:

• NIST standards

• In house Laboratory controls

Measuring the quality and quantity of “DNA “ during the process

Quantity

Qubit® 2.0 or 3.0 (Life Technologies)

Bioanalyzer (Agilent)

Quantitative PCR- initial sample and library quantification

Quality

BioanalyzerIdentifiler and PPY

amplified products

prior to library prep





Control of data



Establishing some starting guidelines for calling alleles

In each sample

> 50 reads must be aligned to a given locus (minimum coverage threshold)

>10 reads or a defined % of the total reads for the locus equivalent to 10 reads was required for an allele to be called

CE based stutter values

Heterozygote locus balance of 50%.

Future Directions

Where to from here....

To implement a specialist service 2 years plus or minusTo implement a routine, all case-sample service 3-5 years

Current areas of development focusAdjusting the testing method to maximise sensitivity for forensic samples

Ensuring reproducibility and reliability

Behaviour of DNA sequences in mixed DNA samples

Software for compatibility with DNA Profiling Databank

Ethical concerns and policy issues

Collection of population data for the New Zealand populations not represented elsewhere

Staff training and development

Current areas of research focus

mtDNA sequencing:

whole mt genome sequencing

extended control region sequencing

Identical Twins

Microbial community sequencing for forensic samples, egsaliva, faeces, skin and vaginal samples

Species identification of non human sample mixtures, Traditional East Asian Medicines and food forensics.

In conclusion:MPS is recognized as a suitable technology for forensic science and is already widely used for clinical applications

MPS is within the capability of most forensic laboratories, given adequate training and resources

Community solutions provide a good entry point for laboratories without R and D options

Custom solutions can provide a more in depth assessment and are also well supported

Acknowledgements

Ryan England – for the laboratory work and most of the data analysis

New Zealand Genomics Limited- initial MPS sequencing services

Ministry of Business Innovation and Enterprise and ESR for research funding

Life Technologies, Melbourne, Australia for assistance with the custom primer panel design.

Life Technologies and its affiliates are not endorsing, recommending, or promoting any use or application of Life Technologies products presented by third parties during this seminar. Information and materials presented or provided by third parties are provided as-is and without warranty of any kind, including regarding intellectual property rights and reported results. Parties presenting images, text and material represent they have the rights to do so.

All products referenced in the presentation are, unless stated otherwise on the individual product labelling, For Research, Forensic or Paternity Use Only. Not for use in diagnostic procedures.

When used for purposes other than Human Identification the instruments and modules of the cited software are for Research Use Only. Not for use in diagnostic procedures.

AGENCOURT AMPURE XP is a registered trademark of Agencourt Bioscience and is for laboratory use only.

Speaker was provided travel and hotel support by Thermo Fisher Scientific for this presentation, but no remuneration

Technology

Massively Parallel Sequencing - integrating the Ion PGM™ sequencer into your forensic laboratory