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Next genera*on mtGenome sequencing for forensic purposes using the Ion Torrent PGM
Dr. Walther Parson
Ins*tute of Legal Medicine, Innsbruck Medical University, Austria walther.parson@i-‐med.ac.at
www.empop.org
Ion Torrent™ Semiconductor Sequencing for Forensic Applications Webinar Feb 14, 2013
Mitochondrial DNA
circular double-‐stranded molecule coding region (15 kb) 37 genes control region (1.1 kb) d-‐loop
evolu?onary rate ~10x of nDNA
(www.mitochondrialdnates?ng.com)
Russell, P. J. (2005)
High mtDNA copy number
Higher copy number than nDNA 4-‐5 mtDNA (avg) molecules/mitochondrion (Satoh and Kuroiwa, 1991) up to 1,000 mitochondria/cell (Robin and Wong, 1988)
MtDNA is maternally inherited
Mitochondria derive from the fer?lized egg – passed along maternal line
(www.accessexcellence.org)
(anthro.palomar.edu)
Applica*on to old historical cases
fibula (Günter Messner) Parson et al 2007
femur (Romanov family) Coble et al 2009
premolar (Wolfgang A. Mozart) Parson 2006
Why mtGenomes?
discrimina?on power, phylogeny, quality control
2011-‐MU-‐MU-‐K402 collabora?on with AFDIL (Dover, DE)
Maximizing mtDNA Tes?ng Poten?al with the Genera?on of High-‐Quality mtGenome Reference Data
Amplicon-‐based sequencing more sensi?ve than compe?tor instruments Amenable to degraded DNA (down to 75 bp) Mul?plex individuals – Barcodes (up to 96) Faster library construc?on Automated enrichment system Very fast sequencing ?me Less total hands-‐on ?me Higher throughput (runs per week) Natural chemistry (less bias, less maintenance)
Why PGM?
42 mtGenomes sequenced with STS and PGM
Samples
Origin # Source Reference
Sub-‐Saharan (Angola) 5 blood Fendt et al 2012
Southeast Asian (East Timor) 8 buccal this study
Westeurasian (Austria) 6 paraffin-‐embedded ?ssue Fendt et al 2011
Westeurasian (Austria) 23 buccal this study
Sanger-‐type Sequencing of mtGenomes
mtGenome PCR with 2 and 9 overlapping amplicons
Sequencing using 106 primers
or
OneTouch™ OneTouch™ES PGM™ Torrent Server & Torrent Browser courtesy Applied Biosystems by Life technologies
PCR e-‐shearing (130-‐140 bp)
100/200 bp chemistry 316 chips
PGM Next Genera*on Sequencing of mtGenomes
BAM (Binary Alignment Map), BAI (Binary Alignment Index) rCRS (mtDNA Reference sequence, Andrews et al 1999) Torrent Browser Variant Caller (Ion Torrent, Life Technologies)
Variant Caller (Vs. 3.2.43647) TMAP Smith-‐Waterman alignment op?miza?on (Li and Homer, 2010)
Integra?ve Genomics Viewer (IGV)
Freeware to visualize alignment files (Robinson et al, 2011; , Vs. 2.1.21 (2541 ) paired read alignment
NextGENe (SokGene?cs)
paired read alignment (Vs. 2.3.1) + visualiza?on Sequencher (GeneCodes)
Tablet for NGS integrated in Sequencher (5.0) GSNAP alignment (Wu and Nacu, 2010)
NGS analysis tools used in this study
Ion Torrent Variant Caller
20% variant frequency threshold (of total coverage) because some low DNA templates were used for NGS
Direct comparison between STS and PGM
PGM seq. chem. # bp differences
100 bp 31 513,651 95 (0.018%)*
200 bp 33 546,786 81 (0.015%)*
16183/4' 16193.XC' 309.XC' 315.1C' 573.XC' Del' Sub'
16183/4'
16193.XC'
309.XC'
315.1C'
573.XC'
Del'
Sub'
16183/4' 16193.XC' 309.XC' 315.1C' 573.XC' Del' Sub'
100 bp 200 bp
* Length heteroplasmy not considered; alignment 5’ (PGM) vs. 3‘ (STS)
HVS-‐2 C tract -‐ 315.1C
PGM #
100 bp 31 (100%)
200 bp 33 (100%)
6C T310 G316
Seems to be an artefact of the TMAP aligner If a modified rCRS with 6Cs between 310 and 316 is used as reference, all 6 C’s are reported by the Ion Torrent variant caller Paired read aligner report the insCs rela?ve to rCRS (see later)
100 bp 200 bp
(rCRS variant) 5C
HVS-‐2 C tract -‐ 309.XC
PGM #
100 bp 16 (51.6%)
200 bp 14 (42.4%)
7C T310
All C-‐tracts between 302 and 310 with the rCRS variant (7 Cs) were concordantly reported between STS and PGM Inser?ons of 1 and 2 Cs (309.1C, 309.2C) were not reported with PGM (TMAP alignment artefact)
100 bp 200 bp
rCRS variant
HVS-‐1 C tract -‐ 16193.XC
PGM #
100 bp 3 (9.7%)
200 bp 5 (15.2%)
4C
T16189
100 bp 200 bp
5C
16183C 16189C 16193.1C
rCRS variant
extensive LHP All rCRS variants concordant
Effect of different aligners on C tract reports
Sanger NextGene - mutation report position score cv 309.1C 310 insC 6,1 130 315.1C 316 insC 2,9 123 16189C 16189 T>C 15,9 208 16193.1C - - -
WGS28
NextGENe IGV Sequencher Tablet
Subs*tu*ons
PGM #
100 bp 29
200 bp 15 100 bp 200 bp
Twenty-‐two (75.9%) differences in 100 bp chemistry caused by 6 samples (not sequenced with 200 bp chemistry due to low DNA amount). Gave also low EPGs for STS.
Majority of subs?tu?on differences in 200 bp chemistry. Some posi?ons were hit in mul?ple samples: 10651 (3), 10664 (3), 2689 (3) lie close to PCR primer binding regions 456 (4) short T-‐stretch between 452-‐455 (alignment)
Dele*ons (outside HVS-‐tracts)
PGM #
100 bp 11
200 bp 13 100 bp 200 bp
A total of 13 (54.2%) reported dele?ons observed together with a subs?tu?on at the same posi?on (the laser confirmed by STS) e.g. WGS05 hg L0d1’2 498del not reported for 200 bp chemistry, present in IGV viewer as 494del
494del
Substan?ally higher through-‐put of mtGenome sequencing with PGM compared to STS High concordance between STS and PGM reports for both chemistry versions (176 differences in 1,060,437 bps -‐ 0.017%; 20% variance call threshold) Roughly two thirds of differences observed in homopolymeric C tracts (> 7Cs); physical PGM sequence data seem to be there, as modified rCRS or other alignment algorithms reveal concordant calls to STS (up to 11 consecu?ve Cs) Some dele?ons outside C tracts observed together with (concordant) subs?tu?ons (alignment algorithm) Sokware constantly improving; TMAP aligner shows generally good performance, paired read aligners seem to perform beser when indels are present in homopolymeric regions with respect to the rCRS
Summary and outlook
Acknowledgements
FP7-‐SEC-‐2011-‐285487
Transla?onal Research project L397 “EMPOP–an innova?ve human mtDNA database”
2011-‐MU-‐MU-‐K402 Maximizing mtDNA Tes?ng Poten?al with the Genera?on of High-‐Quality mtGenome Reference Data
Robert Lagacé Sharon Wooson Reina Langit
Chris?na Strobl Gabriela Huber Bexna Zimmermann Liane Fendt
Samples Sibylle Marcial Gomes, Luis Souto, University of Aveiro, Portugal Rhena Delport, University of Pretoria, South Africa