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HID-Ion Identity Panel - Application Note

HID-Ion Identity Panel - Application Note



The HID-Ion AmpliSeq™ Identity Panel is the first commercially available NGS panel for human identification purposes that provides discrimination of individuals similar to STR genotype match ...

The HID-Ion AmpliSeq™ Identity Panel is the first commercially available NGS panel for human identification purposes that provides discrimination of individuals similar to STR genotype match probabilities used by forensic analysts.

High discrimination power is achieved by using 30 upper Y-Clade SNPs and 90 autosomal SNPs that have high heterozygosity and low Fst, selected from publications by Dr. Kenneth Kidd from Yale University and the SNPforID Consortium.

The panel has been optimized to enable the sequencing of degraded or challenging forensic samples, requiring as little as 1ng of input DNA.




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    HID-Ion Identity Panel - Application Note HID-Ion Identity Panel - Application Note Document Transcript

    • APPLICATION NOTE HID-Ion AmpliSeq™ Identity Panel HID-Ion AmpliSeq™ Identity Panel A community panel designed with leading human identification researchers Identify challenging and degraded samples •• Confidently identify limited, challenging, or degraded samples using 1 tube of primers and 1 sequencing run •• As little as 1 ng of input gDNA required •• Simple Ion AmpliSeq™ PCR-based library amplification Scalability •• Multiplex up to 50 samples per run and decrease your sequencing costs* •• Three Ion Torrent™ chips producing up to 2 Gb of total sequence •• Allows for low-, mid-, and highthroughput lab demands Convenience and speed Introduction Single nucleotide polymorphisms (SNPs) provide a variety of new, important information for human identification purposes, in addition to short tandem repeats (STRs). Due to shorter amplicons required for sequencing, SNPs enable high recovery of information from degraded samples (such as in the case of mass disaster victims). The ability to multiplex hundreds to thousands of SNP primers, and concurrently amplify and sequence the SNPs, provides significant convenience and high discrimination power comparable to the 13 CODIS core STR loci. Additionally, certain SNPs can provide information about a sample owner’s: lineage, ancestry, and external phenotypes (such as hair and eye color). Finally, SNPs have been shown to be helpful in mixture interpretation. Coupled with the simple and scalable Ion Personal Genome Machine™ (PGM™), the HID-Ion AmpliSeq™ Identity Panel has a streamlined workflow that enables users to generate identifying genotypes from degraded or challenging samples in typically 1.5 workdays (Figure 1), using as little as 1 ng of input sample, thereby helping to increase a lab’s success rate. Ion Torrent™ chip options, along with sample barcoding, give each lab the flexibility to determine their preferred number of samples, reads, and coverage performance in each sequencing run while helping to decrease their cost per sample. •• Life Technologies provides an end-to-end solution from sample extraction to postsequencing tools •• Integrated software tools that analyze sequence data (HID SNP Genotyper plugin hosted on Torrent Suite™ Server) •• Go from sample extraction to identifying genotypes in 1.5 days * Sample estimates based on output of Ion™ 318 Chip and number of reads required for optimal performance EXTRACT SAMPLES 0.5 HR 5.5 HR 6.5 HR 4.5 HR 0.5 HR Figure 1. Simple and integrated end-to-end HID-Ion AmpliSeq™ Identity Panel workflow. Users can go from sample extraction to identifying genotypes, typically in 1.5 workdays, using provided reagents, protocols, and easy-to-use software tools.
    • High discrimination power The HID-Ion AmpliSeq™ Identity Panel is one of the first NGS solutions for human identification purposes that provides discrimination of individuals similar to STR genotype match probabilities† used by forensic analysts. High discrimination power is achieved by using 30 upper Y-clade SNPs1 and 90 autosomal SNPs that have high heterozygosity and low Fixation Index (Fst), described in publications by Ken Kidd of Yale University2 and the SNPForID Consortium3 (Figure 2). This end-to-end solution includes the panel of primers and a software plugin tool that runs on the Torrent SuiteTM Server. The software tools distill the sequencing data to identifying genotypes, which can be exported for additional analysis. 48 30 43 30 upper Y-clade SNPs 43 Ken Kidd SNPs 48 SNPforID SNPlex 120 120 SNPs (1 shared SNP) 90 autosomal SNPs Figure 2. SNPs in the HID-Ion AmpliSeq™ Identity Panel. The panel is made up of 90 autosomal and 30 upper Y-clade markers. The Ken Kidd and SNPforID sets share one SNP. HID-Ion AmpliSeq™ Identity Community Panel Targets 120 SNPs (30 Y chromosome, 90 autosomal), including: • 43 unlinked SNPs from Ken Kidd • 48 SNPs from SNPForID Consortium (overlap of 1 SNP with the Ken Kidd set) Average amplicon length 137 bp Primer pools Primer pairs for 120 loci in 1 pool Recommended input DNA 1–10 ng (system will perform with as little as 100 pg but with increased risk for stochastic effects) Amplicon coverage: Number of sequenced reads overlapping a single SNP location (Figure 3) • For 97% of autosomal SNPs to have at least 300x coverage, target an average coverage of 645x • For 97% of autosomal SNPs to have at least 150x coverage, target an average coverage of 261x • This corresponds to ≥66,000 reads per sample on the chip Multiplexing recommended To achieve sufficient amplicon coverage we recommend pooling a maximum of 5, 25, and 50 samples on a single Ion 314™, Ion 316™, and Ion 318™ Chip, respectively † The complete set of 45 unlinked Ken Kidd SNPs has a match probability less than 1 x 10–15 in 44 populations. SNPforID’s 52 SNP-set has a mean match probability of at least 5.0 x 10–19 in nine different populations. Since we use 43 and 48 SNPs (respectively) from these sets, we estimate the match probability of the panel to be ~1 x 10–16 to 1 x 10–18. For comparison, the 13 CODIS core STR loci have a match probability of ~2.4 x 10–15.
    • Verifying the HID-Ion AmpliSeq™ Identity Panel Autosomal amplicon coverage per SNP per individual Genomic DNA (gDNA) was extracted from 32 samples, and their known genotypes (SNaPShot® kit results for Ken Kidd’s 46 SNPs) were compared to results obtained from the panel. Two Ion 318™ chips (20 samples on one chip and 12 on the other) were used for this study. Using software tools (i.e., Torrent Suite™ Server & HID SNP Genotyper 4.0), results from the study demonstrate a concordance rate of 99.71% for the SNPs, at an average read depth of 1,500x per SNP with 10 ng of input per sample. Designed with input from leading researchers: Dr. Kenneth Kidd Department of Genetics Yale University School of Medicine United States Figure 3. Sequenced reads using the HID-Ion AmpliSeq™ Identity Panel sufficiently cover all identifying SNPs. Ten (10) samples were prepared, barcoded, and pooled together into a single template and sequencing run. The chart describes the sequencing coverage for the 90 autosomal SNPs. The x-axis lists the rsSNPIds, the left y-axis shows the coverage of the samples, and the right y-axis (grey bars) measures the strand ratio (where a value of 0 is complete strand bias and 1 is complete strand balance). The dark line across the bars indicates the average coverage of each SNP across the samples. Similar to the Y-SNPs, this panel has minimal variation in coverage (coefficient of variation of 0.26) and high strand balance (strand ratio of 0.88). Dr. Niels Morling Department of Forensic Medicine University of Copenhagen Denmark Figure 4. A sub-panel of the HID SNP Genotyper plugin report.
    • Available to the community The HID-Ion AmpliSeq™ Identity Panel and other community panels may be reviewed and ordered via the Ion AmpliSeq™ Designer at ampliseq.com: 1. Create an Ion Community account by going to the Ion Community, (lifetechnologies.com/ioncommunity). 2. Log in to ampliseq.com using your Ion Community account. 3. Select “Panels tab” and then the “community panels” subtab to review panel design and order. Download the HID SNP Genotyper Plugin at http://ioncommunity.lifetechnologies.com/docs/DOC-3138 “Of interest may also be SNPs for individual identification as they provide strong technical advantages over STRs, such as no slippage artifacts and better ability to deal with degraded DNA due to smaller fragment length.” Dr. Manfred Kayser Department of Forensic Molecular Biology, Erasmus University Rotterdam, The Netherlands References 1. Karafet TM, Mendez FL, Meilerman MB, et al. (2008) New binary polymorphisms reshape and increase resolution of the human Y chromosomal haplogroup tree. Genome Res 18(5):830–838. 2. Pakstis AJ, Speed WC, Fang R, et al (2010) SNPs for a universal individual identification panel. Hum Genet 127:315–324. 3. Sanchez JJ, Phillips C, Børsting C, et al. (2006) A multiplex assay with 52 single nucleotide polymorphisms for human identification. Electrophoresis 27:1713–1724. Ordering information Product Cat. No. HID-Ion AmpliSeq™ Identity Panel Order at ampliseq.com HID PGM™ System (PGM™ + Server) 4476115 Ion OneTouch 2 System 4474779 ™ Ion AmpliSeq Library Kit 2.0 8 reactions 96 reactions 384 reactions 4475345 4480441 4480442 Ion Xpress™ Barcode Adapters Kits 1-96 kit 1-16 kit 17-32 kit 33-48 kit 49-64 kit 65-80 kit 81-96 kit 4474517 4471250 4474009 4474518 4474519 4474520 4474521 ™ Ion PGM™ Sequencing 200 Kit v2 and Template OT2 200 Kit Ion PGM™ Chips (8 pack) 4482006 and 4480974 Ion 314™ Chips Ion 316™ Chips Ion 318™ Chips Please contact your account representative for detailed ordering information. Learn more at http://ioncommunity.lifetechnologies.com/community/applications/hid For Research, Forensic or Paternity Use Only. When used for purposes other than Human Identification the instruments cited are for Research Use Only. Not for use in diagnostic procedures. ©2014 Life Technologies Corporation. All rights reserved. The trademarks mentioned herein are the property of Life Technologies Corporation and/or its affiliate(s) or their respective owners. CO08431 0114 4482261 4483324 4484355