4 Aims, Objectives and Hypothesis
4.3 Hypothesis
By developing a targeted PCR protocol for amplification of five STR regions, and five SNPs for human identification as well as the mitochondrial D-loop before sequencing with the Oxford Nanopore MinIONÔ, we can match a known reference sample to an unknown crime scene sample in an in-field environment.
5 Conclusion
Forensics is a highly regulated field that is cautious to implement new technologies, particularly for DNA analysis; where samples are unable to be reproduced, therefore, effective and accurate analysis is crucial. Extensive research is required before new processes are implemented in standard forensic practice. MinIONÔ has the potential to become a dominant sequencing platform within forensic science, particularly within in-field environments. However, this review has highlighted issues with sequencing errors and data analysis. With continued technological advancement and the development of more user-friendly systems it is expected that these issues can be overcome. Once this occurs research can focus on validating the MinIONÔ system for use in forensic investigation. By continuing research in this field, the current pressures on standard DNA typing procedures can be reduced as DNA evidence can be more frequently employed for human identification in scenarios where forensic DNA analysis would have previously been disregarded.
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7 Appendix
Appendix 1. Core loci utilised by United States, European and Australian government agencies.
Ame lo ge ni n CS F1 PO FG A TH0 1 TP OX
VWA D3S1358 D5S818 D7S820 D8S1179 D13S317 D16S539 D18S51 D21S11 D1S1656 D2S441 D2S1338 D10S1248 D12S391 D19S433 D22S1045 D21S11 D2S1338 SE33 D6S1043 Penta E Penta D US Core Loci US Core Expanded European Core Loci European Expanded Loci Australian Core Loci Australian Expanded Loci
Appendix 2. SNP panel of 19 SNPs for human identification.
Table adapted from Kidd, Pakstis (36). Chromosome Cytogenic
band position Locus symbol ABI catalog # dbSNP rs# Nt. UCSC Position May 2004
ALFRED site
UID FPopulation ST 40 Fpopulation ST 7 Avg. Het. 40 population Avg Het. 7 population
4 p12 GABRA2 C_8263011_10 rs279844 46, 107, 583 S1001391O 0.0302 0.0105 0.485 0.495 13 q32.3 PHGDHL1 C_1619935_1_ rs1058083 98,836, 234 S1001402H 0.0317 0.0135 0.464 0.484 5 q31 SPOCK C_2556113_10 rs13182883 136, 661, 237 S1001390N 0.0333 0.0185 0.471 0.489 1 q21.3-q22 LY9 C_1006721_1_ rs560681 157, 599, 743 S1001392P 0.0345 0.0183 0.434 0.439 10 q26 HSPA12A C_3254784_10 rs740598 118, 496, 889 S1001393Q 0.0403 0.0107 0.463 0.477 6 q22 TRDN C_2140539_10 rs1358856 123, 936, 677 S10014070 0.0400 0.0176 0.473 0.486 18 p11.3 RAB31 C_1371205_10 rs9951171 9, 739, 879 S1001395S 0.0443 0.0196 0.474 0.490 1 p36 PRDM2 C_340791_10 rs7520386 13, 900, 708 S1001394R 0.0452 0.0180 0.477 0.490 6 p24-p22.3 HIVEP1 C_9371416_10 rs13218440 12, 167, 940 S1001397U 0.0466 0.0127 0.457 0.479 6 p24.3 SASH1 C_1256256_1_ rs2272998 148, 803, 149 S1001398V 0.0471 0.0102 0.468 0.490 2 q31.3 CERKL C_1276208_10 rs12997453 182, 238, 765 S1001396T 0.0475 0.0188 0.445 0.466 6 q25 SYNE1 C_251223_10 rs214955 152, 789, 820 S10014031 0.0491 0.0172 0.475 0.491 4 q21.1 RCHY1 C_1880371_10 rs13134862 76, 783, 075 S1OO1400F 0.0537 0.0057 0.456 0.467 10 q23.3-q24.1 SORBS1 C_7538108_10 rs1410059 97, 162, 585 S1001399W 0.0540 0.0120 0.471 0.482 5 qter ADAMTS2 C_3153696_10 rs338882 178, 623, 331 S1001401G 0.0563 0.0186 0.467 0.490 6 q22-q23 THSD2 C_411273_10 rs2503107 127, 505, 069 S1001406N 0.0575 0.0126 0.454 0.463 5 q35 LCP2 C_3032822_1_ rs315791 169, 668, 498 S1001404J 0.0581 0.0176 0.471 0.485 11 q23 KBTBD3 C_1636106_10 rs6591147 105, 418, 194 S1001409O 0.0585 0.0191 0.449 0.481 18 q11.2 B4GALT6 C_7459903_10 rs985492 27, 565, 032 S1001413J 0.0594 0.0149 0.468 0.487
Appendix 3. Information for 52 autosomal SNPs used to generate the SNPforID panel.
NA = Not Available
*lower case letter defines the base replaced with a pyrimidine derivate P base analogue Table adapted from Sanchez, Phillips (39).
Marker Code Chromosome NCBI human
chromosome position NCBI rs No. TSC No. PCR Forward Primer (5’ – 3’) PCR Reverse Primer (3’ – 5’) Amplicon Size TSC allele reported
1 1 4037521 rs1490413 TSC0724193 GTGTGGACTGGGCTGATGT TTCTCACTAGTGTCCCTGCTCTG 68 A/G
2 2 104974 rs876724 TSC0208870 GCAGGCTCCATTTTTATACCACT GAATATCTATGAGCAGGCAGTTAGC 83 C/T
3 3 936782 rs1357617 TSC0496080 AGCTGATGCAGACCACTTCAC GGATAGCTGATAAGAAACATGACCA 90 A/T
4 4 10719942 rs2046361 TSC1065282 CCTATTTGTATGTATCTATTGTCTATGAACG GTCATTGTTGACACTTCACCTTCTA 79 A/T
5 5 2932133 rs717302 TSC0039610 CTTTAGAAAGGCATATCGTATTAACTGTG AACACAGAAAGAGGTTCATATGTTGG 86 A/G
6 6 1080939 rs1029047 TSC0253802 CATAACGTGGATTTGTCAGCA GGAATAAACTGAAGGCTAAAGAAAAG 100 A/T
7 7 4201341 rs917118 TSC0229630 GCCCTTTAGGGTCGGTTC GTAAGAGATGACTGAGGTCAACGAG 87 C/T
8 8 136017 rs763869 TSC0065968 ATCAAGTGCTTTCTGTTGACATTTG GGCTACTCCCTCATAATGTAATGC 100 C/T
9 9 1813774 rs1015250 TSC0097236 AAGTGATGGAGTTAGGAAAAGAACC AAGACATTAGGTGGATTCATAGCTG 95 C/G
10 10 3328178 rs735155 TSC0027519 GGAGAAAACCGGAGAGCTG GAGTGTCACCGAATTCAACG 100 A/G
11 11 11060530 rs901398 TSC0177752 CTGGGTGCAAACTAGCTGAATATC CTGGAATGTACTAGGCAAGAAACTAA 70 C/T
12 12 741262 rs2107612 TSC1108144 GAGCATTCTCTTCTGTTAAAATTGC TGAGTACATTATTCAACTGTTTTGGAG 93 A/G
13 13 20172700 rs1886510 TSC0904551 GTCCTTGTCAATCTTTCTACCAGAG GGATTTTCACAACAACACTTGC 86 C/T
14 14 23840960 rs145361 TSC0684657 AGGGAAATACACCCTGAGCTG AGCTGTCCATCATCAGTAAGACAC 73 A/T
15 15 22119157 rs2016276 TSC0326290 TGCATCCCAGCCTCCACT ATTGTACCTTGCCACTTTGTGTG 90 A/G
16 16 5606490 rs729172 TSC0028090 CATTAATATGACCAAGGCTCCTCT ACATTTCCCTCTTGCGGTTAC 60 A/C
17 17 5907188 rs740910 TSC0105771 GTATAACAGTTTGCTAAGTAAGGTGAGTG AGATAGGTTCGAGTTTTGGCTTTA 87 A/G
18 18 1117986 rs1493232 TSC0729796 CTATTCTCTCTTTTGGGTGCTAGG CAAACTGTTTATTGTGAGGCCTGT 59 C/A
19 19 33155177 rs719366 TSC0044247 CCACAGCATCTTTTAACTCTTTTATTATCC GTAAGGACTTATAGTGAGTAAAGGACAGG 105 C/T
20 20 4442483 rs1031825 TSC0334834 CTTATCTTTCCCACATTATGGTCCT AAGATATAATCACTGCTTTCAAGTATGC 98 A/C
21 21 15607469 rs722098 TSC0050288 GGAAGTACACATCTGTTGACAGTAATGA GGGTAAAGAAATATTCAGCACATCC 81 A/G
22 22 26141338 rs733164 TSC0023085 AGCTTTCAGCCCCAGGTC CGGCTCAGGAATGTCAGG 68 A/G
23 10 2360631 rs826472 TSC0557086 TGAATTTTGTCTCTGTTATATTAGTCACC TGTAATTGAAATTTGTAGGCAATAGAC 85 C/T
24 21 28601558 rs2831700 NA GGCTAAACTATTGCCGGAGA TTCCCTAGAACCACAATTATCTGTC 62 G/A
25 14 96835572 rs873196 TSC0202434 GCATTCAAATCCCAAGTGCT GCAGGAGTTGGAGTCAATCAG 63 C/T
26 16 79885888 rs1382387 TSC0544547 ACGAAGGAGAAACACCTGAACT TGGAGTACTTAATAAGACGCTGCAT 69 G/T
27 12 104830721 rs2111980 TSC1113476 AGCATCTTGGCAGCATCC AGCAAGATCTTTGCCAGTGAGT 72 A/G
28 8 139370172 rs2056277 TSC1082757 CCAAACTGGGTGTTAGGGAGAC TCATTATCTCGTCATACTTCCCTGT 73 C/T
29 18 73559363 rs1024116 TSC0247167 CCATGTGTTCTAATAAAAAGGATTGC TGGGAAGTGAGCAAAAGTAAATACA 76 A/G
30 6 164954622 rs727811 TSC0062764 GTGTTTCTTTTTCTCTTACCGGAAC GTGAATGAAATCATGAGATTGCTG 78 A/C
32 1 239753521 rs1413212 TSC0607362 AACCTCCTTTGGAAACACTGAC CAACATTCCATTATCCAGGAGAC 84 A/G
33 17 78065617 rs938283 TSC0357388 CATTGAAGTCCTAACCCCTAGTACG GGATGAGGCCCACCCATA 85 C/T
34 4 191013970 rs1979255 TSC0925231 TCAGAGACTATGGATGGTATTTAGGTC CATGGAACGTTGGAACTCTTG 86 C/G
35 9 122257493 rs1463729 TSC0377760 ACTATCAGTCTCTGCCCTTATTCTG CACATGTGCATGCTTTTGG 87 A/G
36 11 134205198 rs2076848 TSC0022275 GCCTCACCACCAGAAATCAG GACATCAGAAACTCCCATGAAACT 89 A/T
37 3 192127021 rs1355366 TSC0491662 CCATGATTTTCTTGTGGTGAGA CACATGTGCTTAGGCCACAAC 90 A/G
38 2 239850329 rs907100 TSC0186810 GGAGTTCCTGATAACGATTCTGAAG ACAGAAAAGAAGCcCAGTTGGA* 91 C/G
39 13 104636412 rs354439 TSC0700528 GGCTTCTCTTTCCCTTATGTATCTC CAGGTTGCGATAGAAAACAGTGAAT 93 A/T
40 22 46048653 rs2040411 TSC1056845 TCTGGAATGCCAGTTCTTTTGT CAGAACGCCTATGAAAACCAGT 94 A/G
41 7 154850085 rs737681 TSC003074 ACATGTGAGGCCATCTCCAC CCTTACTGTGATGTAGGCACTGTTC 96 C/T
42 21 27530034 rs2830795 NA CACTTCTATAGACATAGGACACACCAT ATCTAGGCTCTGAATCAGGATGAG 97 A/G
43 5 174759601 rs251934 TSC0220872 AGAGGGCAGTGAGGCTTTTAAGTAG TGCTAGAATCCAGACTTAACTACCAG 98 C/T
44 21 41336325 rs914165 TSC0197658 AGCAGCAGAGCCTGGATG AGACCAGTCACCTCTTTTGCACT 100 A/G
45 1 235480457 rs10495407 NA AGATCTCCACTTCCTCTTGGTTG CTCCCAAATTTACATTGCCACT 102 G/A
46 9 1234344108 rs1360288 TSC0501229 AGACTCTCTGTGTGTGGCTTTG GAGGGGGCATCTGTTGAG 103 C/T
48 10 132172819 rs964681 TSC0270699 GTACCTGGAGGTGATTTCTGTGAG GTTATGGAGGATTGGTAAGAACCAG 106 C/T
49 20 40172539 rs1005533 TSC00802071 GGTTTGTGTGTGAGTGTTTCAGAT CCTTATGCCTCCCCTGAAC 107 A/G
50 15 51332965 rs8037429 NA TTCACTTTGCTACACCTCCATAGTA TGCTACGTAAGAGGTCATTGCTATC 108 C/T
51 1 236923075 rs891700 TSC0162577 TTTTCAGAGGTGGTATTTCTAGCTG GCTATGACACTCCTTAGAACTATGCAA 109 A/G
52 13 18699724 rs1335873 TSC0829150 GTGGATGATATGGTTTCTCAAGG TTCAACAAACGTGTGATGCTCT 110 A/T
53 22 46574531 rs1028528 TSC0253071 ACAGCTGATGCCTCCCTGA GAGGATGAAGGTTAGAGCCAGAC 113 A/G
Part Two
Manuscript
TARGETED STR AND SNP IN-FIELD SEQUENCING BY OXFORD NANOPORE
MINIONÔ FOR THE IDENTIFICATION OF AN INDIVIDUAL IN A MILITARY
Abstract
DNA sequencing is a valuable tool within forensics. Traditionally DNA sequencing is conducted in a centralised laboratory that requires the use of specialised equipment and highly trained individuals to interpret the results, which is known to be costly and time consuming. The demand for DNA sequencing within forensic is increasing, prompting demand for improved sequencing technologies. Oxford Nanopore Technologies released the MinIONÔ sequencer, a portable sequencing platform that is able to discriminate single molecules in real time. The research presented here aimed to develop a targeted PCR amplification approach for STRs, SNPs and mtDNA before sequencing with MinIONÔ to allow for rapid identification of an individual in an in-field environment. The use of a targeted approach to isolate regions of forensic interest was demonstrated; however, the method presented here has not superseded current forensic DNA typing methods. It does however demonstrate the potential of in-field sequencing platforms for use in forensic applications.
Keywords
Table of Contents
Abstract ... 43 Keywords ... 43 List of Figures ... 46 List of Tables ... 46 Appendix ... 46 List of Abbreviations ... 46 1. Introduction ... 48 2. Materials and Methods ... 51 2.1. Target Selection ... 51 2.2. Research Strategy ... 51 2.3. Primer Design ... 52 2.4. Sample Collection ... 53 2.5. DNA Extraction ... 53 2.6. PCR Amplification ... 53 2.7. PCR Clean Up ... 53 2.8. Fragmentation of 2800M Genomic Control DNA ... 54 2.9. DNA Quantification ... 54 2.10. End Repair ... 54 2.11. Ligation to 2800M Control DNA ... 54 2.12. Library Preparation ... 55 2.13. Nanopore Sequencing with MinIONÔ ... 55 2.14. Data Analysis ... 56 3. Results and Discussion ... 57 3.1. Target Selection ... 57 3.2. Primer Design ... 57 3.3. Species Determination of Sequenced DNA ... 59 3.4. Isolation of Targets from Sequencing Data ... 60 3.5. Analysis of STRs and Determination of Genotype ... 61 3.6. Comparison of MinIONÔ Generated STR Profile to Known Profile ... 633.7. Analysis of SNPs ... 64 3.8. Analysis of mtDNA ... 68 3.9. Limitations ... 71 4. Conclusion ... 72 5. References ... 73 6. Appendix ... 77
List of Figures
FIGURE 1. DIAGRAMMATIC REPRESENTATION OF THE SCIENTIFIC STRATEGY FOR THIS RESEARCH. ... 52 FIGURE 2. PERCENTAGE CHARACTERISATION OF (A) FORENSIC SAMPLE AND (B) REFERENCE BUCCAL FROM WIMP ANALYSIS. ... 60 List of Tables
TABLE 1. PRIMER SEQUENCES FOR THE SELECTED SNPS, STR AND MITOCHONDRIAL REGIONS ... 58 TABLE 2. TOTAL READS FOR THE REFERENCE BUCCAL AND THE FORENSIC SAMPLE AND CHARACTERISATION OF TOP FOUR GENUS BY NUMBER OF READS BY WIMP ANALYSIS. ... 60 TABLE 3. STR GENOTYPES. CHARACTERISED ALLELES FOR THE FORENSIC SAMPLE AND REFERENCE BUCCAL. THE
KNOWN PROFILE WAS OBTAINED FROM A COMMERCIAL STR KIT (GLOBALFILER). ... 66 TABLE 4.SNP POLYMORPHISMS IDENTIFIED IN THE FORENSIC SAMPLE AND THE REFERENCE BUCCAL. EXPECTED POLYMORPHISMS AND NUCLEOTIDE POSITIONS OBTAINED FROM DSSNP DATABASE. ... 67 TABLE 5. MTDNA SNP POLYMORPHISMS IDENTIFIED IN THE FORENSIC SAMPLE AND REFERENCE BUCCAL.
POLYMORPHISMS OBTAINED FROM MITOMAP DATABASE. ... 70 Appendix
APPENDIX 1. PRIMER PROPERTIES ... 77 List of Abbreviations a-HL a-Hemolysin bp base pairs CE capillary electrophoresis HVR1 hypervariable region 1 HVR2 hypervariable region 2 IDT Integrated DNA Technologies mRNA messenger RNA
mtDNA mitochondrial DNA
NCBI National Centre for Biotechnology Information NEB New England Biotech
NGS next-generation sequencing
NIST National Institute of Standards and Technology nuDNA nuclear DNA
PCR polymerase chain reaction
rCRS revised Cambridge Reference Sequence SNP single nucleotide polymorphism
STR short tandem repeat
ONT Oxford Nanopore Technologies WIMP What’s in my Pot
1. Introduction
Since the development of Sanger Sequencing by Fredrick Sanger in 1975, the discipline of genomics has undergone vast development (1). Also referred to as first-generation sequencing, these initial sequencing platforms aided many developments within biological science (1). However, these methods are limited by low throughput and high costs. Advancements in polymerase chain reaction (PCR) and recombinant DNA technologies as well as the demand for more affordable, faster sequencing technologies led to the introduction of second-generation platforms (1). Also referred to as next-generation sequencing (NGS) these platforms include the Roche 454 pyrosequencing, Illumina/Solexa, ABI SOLiDÒ, and Thermo Fisher Ion TorrentÔ (2). These systems, particularly the Illumina platform, have had a dominant position in the genomics market as they can provide faster and cheaper results than their first-generation counterparts (1-3). However, they are limited by short read lengths and fragmented genome assemblies, prompting the development of third-generation sequencing technologies (1).
Third generation platforms offer increased read length, a reduction in sequencing time and the biases introduced by PCR amplification (1). The most notable third-generation technology is nanopore sequencing (2). Nanopores allow for a single-molecule sequencing approach (4). Biological nanopores are a protein channel that is formed in a lipid bilayer while solid-state nanopores are synthetically manufactured by drilling or etching into a solid-state