Estudios de marcadores STR por MPS · 2020-06-17 · Estudios de marcadores STR por MPS . para uso...
Transcript of Estudios de marcadores STR por MPS · 2020-06-17 · Estudios de marcadores STR por MPS . para uso...
Estudios de marcadores STR por MPS para uso forense: Validación de kits y Estudio Poblacional Español
Madrid, 28 de Mayo de 2019
Pedro A. Barrio1, Pablo Martin1, The DNASEQEX Consortium, Antonio Alonso1
1 Servicio de Biología del Instituto Nacional de Toxicología y Ciencias Forenses (INTCF), Departamento de [email protected]
Massive Parallel Sequencing (MPS). Paradigm shift
CE (Sanger) MPSvs
DNA-STR Massive Sequencing & International Information ExchangeHOME/2014/ISFP/AG/LAWX/4000007135
Comparison of two MPS platforms: Ion S5 (Thermo Fisher Scientific) MiSeq FGxTM (Illumina)
Madrid Innsbruck Berlin
STRs Standardization typing by MPS
International Exchange of MPS data
Population Studies
Main Applications of MPS
Identification (forensic casework)
Cases of complex relationships and / or family searches
Mixtures ResolutionDegrade DNALTDNA
Missing Persons and Mass Disasters Investigations (DVI)
European Survey on Forensic Applications of MPS
Alonso et al., FSI:G (2017), 29:e23-e25
33 ENFSI DNA WG laboratories from 25 countries
Participation: 54% of all ENFSI DNA Labs(53 Member Institutes and 8 Associated Members)
52%48%
Does your laboratory have a Massively Parallel Sequencing instrument?
YESNO
61%
39%
Does your laboratory have a Massively Parallel Sequencing instrument or Will your laboratory purchase a MPS instrument in the next 18
months?
Yes
No14
119
118 8 8
3 3 2
19 18 1715 15 14
118 8
3
Please check for which category or categories of Forensic DNA markers MPS iscurrently used or being evaluated in your lab and for which ones will be developed in
the next 18 months
Number of labs in December 2016 Number of labs in 2018
> 75%
Outline
Kits ValidationEarly Access Applied Biosystems Precision ID Globalfiler Mixture ID™ PanelEarly Access Applied Biosystems Precision ID Globalfiler NGS STR Panel for the Ion S5™
Spanish Population StudyMüller et al., FSI:G (2018), 36:95-103
Material: Panels
Early Access Precision ID GlobalFiler® Mixture ID Panel
STRs29 autosomal STRs
1 Y-STRsSNPs
42 autosomal2 Y chromosome
InDelsAmelogeninY-InDel rs2032678
Microhaplotyes36 clusters (2-4 SNPs)
Early Access Precision ID GlobalFiler® NGS STR Panel
STRs29 autosomal STRs
1 Y-STRsInDels
AmelogeninY-InDel rs2032678
Material: Panels
Ion Chef
Methods: Analytical phase. Workflow
Ion Chef Torrent Server & Torrent BrowserS5 / S5xl
Samples Type
NIST 2391_A_female sNIST 2391_B_male sNIST 2391_C_male sNIST 2391_D_mixture mNIST 2391_E_female sNIST 2391_F_male s
NIST 2372_A_male s
9947A_female s2800M_male s007_male s
GEDNAP2012_44_S3 sGEDNAP2012_45_S2.1 sGEDNAP2013_46_S2.1 sGEDNAP2013_46_S3.1 sGEDNAP2013_47_S1 sGEDNAP2013_47_S2 sGEDNAP2014_48_S3 sGEDNAP2014_48_S4 sGEDNAP2015_49_S4.1 sGEDNAP2015_50_S2 sGEDNAP2015_50_S3.1 sGEDNAP2015_51_S4 s
GEDNAP2012_44_S4 mGEDNAP2012_45_S1 mGEDNAP2012_45_S3.1 mGEDNAP2013_46_S1 mGEDNAP2013_47_S3 mGEDNAP2013_47_S4 mGEDNAP2014_48_S1 mGEDNAP2014_48_S2 mGEDNAP2015_49_S1 mGEDNAP2015_49_S3 mGEDNAP2015_50_S1 mGEDNAP2015_50_S4 mGEDNAP2015_51_S3 m
GHEP2013_M4.2 sGHEP2015_M6 mGHEP2016_M7.2 m
PS_MADRID_S1 sPS_MADRID_S2 sPS_MADRID_S3 s
TOOTH sFEMUR_NEONATAL sPARAFFIN_BLOCK sPARS_PETROSA s
Stantard Reference Material (Certificated)
Reference Material (NO Certificated)
Proficiency Exercises Samples (GEDNAP)
Proficiency Exercises Samples (GHEP)
Reference Material - Project Staff
Casework-type Samples
EA Precision ID GlobalFiler® Mixture Panel
EA Precision ID GlobalFiler® NGS STR Panel
Sensivity Coverage Study Stutter Study ConcordanceSingle Profiles Mixed Profiles
Material & Methods
Total Replicates Analyzed: 56 (GFm); 106 (GF)
- GlobalFiler®
Previously typing by CE (ABI3500)
- PowerPlex® Fusion 6C
Samples (N = 43)
Results. Some Forensic Parameters Analyzed
Sensitivity:# Markers Replicate_1 Replicate_2 Replicate_1 Replicate_2 Replicate_1 Replicate_2 Replicate_1 Replicate_21 AMELX2 AMELY3 CSF1PO4 D10S1248 I I5 D12ATA63 A A/I6 D12S391 I A A A7 D13S317 A8 D14S14349 D16S539
10 D18S51 A A A A/I A A11 D19S433 A A12 D1S1656 A A A A A A A13 D1S1677 A I I14 D21S11 A15 D22S1045 A16 D2S1338 A A A17 D2S44118 D3S1358 I I19 D3S4529 A A A A A A/I A20 D4S240821 D5S2800 A/I A I22 D5S818 A I23 D6S1043 A24 D6S474 A A/I A A25 D7S820 I I26 D8S1179 A I27 DYS391 A28 FGA A A A A I29 TH01 I I30 TPOX A31 vWA I I
24 cycles
500 pg (250 pg) 250 pg (125 pg) 125 pg (62 pg) 62 pg (31 pg)Control SRM 2372_A
32 samples (2 controls / 8 serial dilutions / 2 replicates)
Stutter Ratios:
0,000
0,050
0,100
0,150
0,200
0,250
0,300
0,350
D2S1
776
TH01
D4S2
408
D3S4
529
D5S2
800
D2S4
41TP
OX
DYS3
91D7
S820
D6S4
74D1
3S31
7CS
F1PO
D21S
11D8
S117
9D2
S133
8D1
S167
7D1
4S14
34D3
S135
8D1
8S51
D5S8
18D1
9S43
3D6
S104
3vW
AFG
AD1
0S12
48D1
6S53
9D2
2S10
45D1
S165
6D1
2S39
1D1
2ATA
63 All
Stutter -4 by STRs markers
Stutter Thresholds:0,000
0,100
0,200
0,300
0,400
0,500
0,600
D2S1
776
TH01
D4S2
408
D3S4
529
D5S2
800
D2S4
41TP
OX
DYS3
91D7
S820
D6S4
74D1
3S31
7CS
F1PO
D21S
11D8
S117
9D2
S133
8D1
S167
7D1
4S14
34D3
S135
8D1
8S51
D5S8
18D1
9S43
3D6
S104
3vW
AFG
AD1
0S12
48D1
6S53
9D2
2S10
45D1
S165
6D1
2S39
1D1
2ATA
63 All
Stutter -4 ratio Threshold (media + 3SD) by STRs markers
23 single-source samples, ≈ 1 ng of DNA input
Concordance
Samples TypeQ
(ng/µL)
AMEL
X
AMEL
Y
CSF1
PO
D10S
1248
D12A
TA63
D12S
391
D13S
317
D14S
1434
D16S
539
D18S
51
D19S
433
D1S1
656
D1S1
677
D21S
11
D22S
1045
D2S1
338
D2S4
41
D3S1
358
D3S4
529
D4S2
408
D5S2
800
-250
0
D5S8
18
D6S1
043
D6S4
74
D7S8
20
D8S1
179
DYS3
91
FGA
TH01
TPO
X
vWA
NIST 2391_A_female s C C C C CI CI C C C CA C CA CI C C CA C-IS C C C C C C C C C CA CA C C C
NIST 2391_B_male s C C C C CI C C C C CA CI C C C CI C C C C C C CI CI C C C C CA C C CI
NIST 2391_C_male s C C C C C CA C C C CA C C CI C C C C C C C C-IS C C C C C C CII CI C C
NIST 2391_D_mixture m C C CI CI C AI C C C CA CI AI C CI C CA C-IS CI C C C-IS C CI C CI CI C CII C CI CI
NIST 2391_E_female s C C C C CI CI C C C C C CA CI C CI C C-IS C C C C CI C C CI C C CI C C C
NIST 2391_F_male s C C C C C C C C C CA C CA C C C C C C C C C C C CI C C C CA C C C
NIST 2372_A_male s C C CI CI C CI C CI C C CI C CII C C-(IS) C CI C-IS C CI CI C CI
9947A_female s C C C C C C C C C C C CI CI C CI C C C-IS C CI CI C C
2800M_male s C C C C C C C C CA C C C C C C C C C C CA C C C
007_male s C C C C C C C CA C C C CI C C C C C C C CA C C CI
GEDNAP2013_46_S2.1 s 0.62 C C C CI C C CI C CI C CI CI C CI C CI CI CI C C C C C
GEDNAP2013_46_S3.1 s 0.6 C C C CI C CA CI C CI CI C C CA CI C CI CI CI C CI CI CI C
GEDNAP2013_47_S1 s 0.4 C C C C C C C C C CI CI C C C C C C C C C C C CI
GEDNAP2013_47_S2 s 2.38 C C C C C C C C C C C CI C C C C C C C CA C C C
GEDNAP2014_48_S3 s 2.9 C C C C CII C C C C C C C C C C C C CI C C C C C
GEDNAP2014_48_S4 s 1.16 C C C C CII C C C CI C CI C C C C C C C C C C C CI
GEDNAP2015_49_S4.1 s 15.6 C C CI C C C C C C C C C C C C C C C C C C C C
GEDNAP2015_50_S2 s 0.62 C C CI CI CI CI C C C C CI C CI C AO C C C C C C C C
GEDNAP2015_50_S3.1 s 4.20 C C CI CI CII C C C C CI C C C C C C C C C C C C C
GEDNAP2015_51_S4 s 1.38 C C CI C CA CI C C C C C C C C CI C C C C C CI C C
GHEP2013_M4.2 s 0.44 C C C C CA C C C CI C C C CI C C C C C C C C C C-IS
GEDNAP2012_44_S4 m 2 C C C C C-IS CI C C-IS C-IS C-IS C CI CI CI C-IS CI C C C C C CI C
GEDNAP2012_45_S1 m 3 C C CI C C C C C CI C CI C CI C CI CI C C C C C CI C
GEDNAP2012_45_S3.1 m 3 C C C C C C C CI CI CI-IS CI CI C C C-IS CI C C-IS C C C C C
GEDNAP2013_46_S1 m 1.61 C C C CI C CI CI C CI CI-IS C-2IS CI C CI C-3IS CI CI CI C C C CI C-IS
GEDNAP2013_47_S3 m 1.71 C C CI CI AOA-IS C CI CI AOI AO AOI CI C C C CI C AO C AO CI AOI C
GEDNAP2013_47_S4 m 1.7 C C C CI C C C C C C C C C CI C C CI C C C C C C
GEDNAP2014_48_S1 m 3.44 C C C C CI CI CI CI C C C-IS CI C CI C CI C CI C C CI C C
GEDNAP2014_48_S2 m 1.93 C C C C CI CI C CI C C CI CI CI CI C CI CI CI C CI CI CI C
GEDNAP2015_49_S1 m 2.58 C C CI CI CI C C CI CI C-IS C-IS C C C C CI C C-IS C C C C C
GEDNAP2015_49_S3 m 4.5 C C CI CI CI CI CI C C CII C C C CI C-IS C C C C C C C CA
GEDNAP2015_50_S1 m 2.8 C C C C CA CI C C C C C CI C C C-IS C CI C C C CI CI CI
GEDNAP2015_50_S4 m 6.74 C C CI C AOI CI C C CI C CI CI C C C-IS CI CI C C C C C CI
GEDNAP2015_51_S3 m 1.14 C C CI C CI CI CI C CI AOI CI CI CI CI CI-IS CI CI CI C C CI CI C
GHEP2015_M6 m 2.28 C C C C C C C C-IS CI CI CI CI CI C-IS CI C CI C-IS C C CI C C-IS
GHEP2016_M7.2 m 1099 C C CI CI AOI CI CI AOI C CI CI CI C C CI CI CI CI C CII CI AOI CI
PS_MADRID_S1 s 23.13 C C CI C C C CI CA C C C CI C C C C C C C C C C CI
PS_MADRID_S2 s 144.59 C C C CI CI C C CA C C C C C C C C C C C CA C C C
PS_MADRID_S3 s 1.84 C C C C C CI C CA CI C C-IS C C C C C C C C CA C C C
TOOTH s 0.019 C C C C C C C C C CI C C C C-IS CI C C CI C CA C CI C-IS
FEMUR_NEONATAL s 0 C C C CI CI C CI CI C AO-IS C CI C-D C CI C CI C-IS C CI-D C C CI
PARAFFIN_BLOCK s 0.0154 C C CI C CI C C C C CI C CI CI C C C C C C C C C C
PARS_PETROSA s 0.0246 C D 11,12 C-D C-D CI-D 11,12 14,(19) CA-D C-D C-D D DA C-D CI-D C-D 10,11,12-D C-D C C CI 11 C-D
Markers / Locus
Reference Material
Casework-type Samples - Singles
Casework-type Samples - Mixtures
Reference Material - Project Staff
Casework-type Samples
% Concordance
Single-source: 99.90 %(1 drop-out / 1046 alleles)
Mixed-source: 98.91 %(11 drop-outs / 1011 alleles)
27 single samples 16 mixtures
Samples (N = 43) Total Replicates Analyzed: 106
62 pg (up to 31 pg with some losses)
on average <15%
on average <30%
Results. EA Precision ID GlobalFiler® NGS STR Panel
Some Details… NIST 2391_A
Isoalleles(isometric heterocygotes)
% Loci with isoallelesSingle-source: 1.53 % (8/523)
Mixed-source: 7.94 % (25/315)
Results. EA Precision ID GlobalFiler® NGS STR Panel
Casework… Pars Petrosa
QFDUO Total: 0.025 ng/µLQFDUO Male: 0 ng/µLIPC: 29.75
Inter-Laboratory Evaluation Study
Innsbruck (GMI)
Interlocus balanceStutter analysis
Sensitivity
Madrid (INTCF)
EA Applied Biosystems Precision ID Globalfiler Mixture IDTM & Globalfiler NGS STR Panels
for the Ion S5TM
Müller et al., FSI:G (2018), 36:95-103
Results. EA Precision ID Globalfiler® Mixture Panel
Interlocus Balance
Interlocus balance varies from 4.7 % (D22S1045) to 241.4 % (TH01) compared to the calculated expected value (100 %)
Results. EA Precision ID Globalfiler® NGS STR Panel
Interlocus Balance
Interlocus balance varies from 22.3 % (D22S1045) to 182.7 % (TH01) com-pared to the calculated expected value (100 %)
Results. EA Precision ID Globalfiler® Mixture Panel
Stutter Analysis
Stutter ratios range from 6.5 % (TPOX) to 24.1 % (D22S1045)
Dataset includes single person samples (1 ng DNA input), homo- and heterozygous genotypes as well as isometric allele calls
Results. EA Precision ID Globalfiler® NGS STR Panel
Stutter ratios range from 5.6 % (TH01) to 18.9 % (D12ATA63)
Stutter Analysis
Dataset includes single person samples (1 ng DNA input), homo- and heterozygous genotypes as well as isometric allele calls
Sensitivity: NIST 2372_A (& Valuation of Cycles Increment)
Results. EA Precision ID Globalfiler® NGS STR Panel
24 PCR cycles
Standard PCR conditions display the expected decrease in reads/marker relative to DNA input
28 PCR cycles
Changed cycling conditions affect marker balance
15 µL 7 + 7µL
+
Sensitivity: NIST 2372_A
# Markers Replicate_1 Replicate_2 Replicate_1 Replicate_2 Replicate_1 Replicate_2 Replicate_1 Replicate_21 AMELX2 AMELY3 CSF1PO4 D10S1248 I I5 D12ATA63 A A/I6 D12S391 I A A A7 D13S317 A8 D14S14349 D16S539
10 D18S51 A A A A/I A A11 D19S433 A A12 D1S1656 A A A A A A A13 D1S1677 A I I14 D21S11 A15 D22S1045 A16 D2S1338 A A A17 D2S44118 D3S1358 I I19 D3S4529 A A A A A A/I A20 D4S240821 D5S2800 A/I A I22 D5S818 A I23 D6S1043 A24 D6S474 A A/I A A25 D7S820 I I26 D8S1179 A I27 DYS391 A28 FGA A A A A I29 TH01 I I30 TPOX A31 vWA I I
24 cycles
500 pg (250 pg) 250 pg (125 pg) 125 pg (62 pg) 62 pg (31 pg)Control SRM 2372_A
Replicate_1 Replicate_2 Replicate_1 Replicate_2 Replicate_1 Replicate_2 Replicate_1 Replicate_2 MarkersAMELXAMELY
A I CSF1POI A D10S1248
I I I D12ATA63I D12S391
D13S317I A A D14S1434I D16S539
A D18S51D19S433
I I D1S1656I D1S1677
A A D21S11D22S1045
A D2S1338I I I D2S441
I D3S1358I I A D3S4529
D4S2408D5S2800
A A D5S818A D6S1043A D6S474
I D7S820I D8S1179
DYS391I A/I FGA
I TH01TPOX
I I vWA
28 cyclesControl SRM 2372_A
125 pg (62 pg) 62 pg (31 pg) 31 pg (15 pg) 15 pg (7 pg)
A I A/I A I A/I A
Com
plet
e
Com
plet
e w
ith A
rtef
acts
Com
plet
e w
ith
Imba
lanc
e >3
0%
Com
plet
e w
ith A
rtef
acts
an
d Im
blan
ce >
30%
Drop
-out
Drop
-out
and
Art
efac
ts
Drop
-out
and
Imba
lanc
e >3
0%
Drop
-out
and
Art
efac
ts
and
Imba
lanc
e >3
0%
Locu
s Dro
p-ou
t
Locu
s Dro
p-ou
t and
Ar
tefa
cts
LEGEND
(& Valuation of Cycles Increment)
Results. EA Precision ID Globalfiler® NGS STR Panel
* Comparable results between INTCF and GMI in terms of:Interlocus balanceStutter ratios
* Minimal handling (IonChef / Ion S5), Complete Integration with forensic LIMS system
* High number of samples / markers analyzed at the same time
Inter-Laboratory EvaluationConclusions
* Sensitivity:Fully automated sensitivity testing yielded 98.6 % alleles down to125 pg (Globalfiler® NGS STR Panel) >> up to 62 pg… or even up to 31 pg (with some losses)
* Concordance:367/376 alleles (97.6 %) concordant to known reference (Globalfiler® Mixture Panel)
376/376 alleles (100 %) concordant to known reference (Globalfiler® NGS STR Panel)
* Increase in number of different alleles: Isoalleles (sequence alleles – isometric heterocygotes)
Loci with more Isoalleles: D3S1358, D8S1179, D1S1656, D21S11, D2S441, vWAVery useful in the case of mixtures, or even kinship investigation
* Works relatively well on Degraded or Low Template DNA samples (LTDNA)
Outline
Kits ValidationEarly Access Applied Biosystems Precision ID Globalfiler Mixture ID™ PanelEarly Access Applied Biosystems Precision ID Globalfiler NGS STR Panel for the Ion S5™
Spanish Population Study
Barrio et al., FSI:G (2019)manuscript in review
498 samples
Material & Methods
Spanish ancestries representing all the 17 Autonomous Communities of Spain (i.e. “regions”)
Concordance Study CE/MPS221 samplesPowerPlex Fusion 6C System (Promega, Madison, WI, USA)
Precision ID GlobalFiler® NGS STR Panel v.2
STRs29 autosomal STRs
Material: Panels
Aditional markers (v.2)Penta DPenta ESRY
1 Y-STRsInDels
AmelogeninY-InDel rs2032678
A total of 31 auSTRs
Methods: Analytical phase. Workflow
Ion Chef Torrent Server & Torrent BrowserS5 / S5xlManually
Methods: Data Analysis
1. Torrent Server Suite (TSS), v. 5.6.0
2. Converge, v. 2.0
3. Discordances Analysis
4. Statistic Analysis
0
50
100
150
200
250
300
350
400
450
500
13.1452
14366
Dept
h of
Cov
erag
e
Alleles
Sample M17-06603-60-608-EX-1_D2S441
STRait Razor v3 (Woerner et al., 2017)
updated Forensic STR Sequence Guide v4 (Phillips et al., 2018)
(Gouy et al., 2017)
IGV v2.4.16 (Thorvaldsdottir et al., 2013)
Results. Sequencing results
Overall coverage data by locus
Results. Concordance Study
Putative mismatches:3 loci:
5 samples: 5 samples out of 221 (97.73 % sample concordance)
1 locus/sample: 5 loci out of 5083 (99.90 % locus concordance)
1 allele/locus: 5 alleles out of 10166 (99.95 % allele concordance)
Penta D, D2S441 and D19S433
Results. Allele frequencies
STR allelic gains by sequence:
Number of alleles compared to heterozygosity observed for the 31 auSTR loci
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
0,600
0,620
0,640
0,660
0,680
0,700
0,720
0,740
0,760
0,780
0,800
0,820
0,840
0,860
0,880
0,900
0,920
0,940
0,960
0,980
1,000
Lenght Repeat Region Sequence Flanking Region Sequence
accession number STR000248
Results. Allele frequencies
SNPs in the flanking regions:
Results. Population genetics
Decrease of Match Probability (PM) (length-sequence)
0,0000
0,0100
0,0200
0,0300
0,0400
0,0500
0,0600
0,0700
0,0800
0,0900
20 CODIS core STR loci: 1.999E-24 (length-based)
31 auSTR loci: 14.828E-40 (sequence-based)
>> 1.043E-27 (sequence-based)
0,0000
0,5000
1,0000
1,5000
2,0000
2,5000
3,0000
3,5000
4,0000
4,5000
Results. Population genetics
Increase of Typical Paternity Index (TPI) (length-sequence)
20 CODIS core STR loci: 1.420E+08 (length-based)
31 auSTR loci: 7.850E+13 (sequence-based)
>> 7.483E+09 (sequence-based)
* There is substantial variation within repeat motifs and/or their immediate flanking regions of a number of STR markers.
Conclusions. Highlights
* MPS allows to analyze a high number of samples/markers at the same time.
* The knowledge of the MPS sequence demonstrated that some of the micro-variantswere erroneously called by CE >> alleles with complete repeat units plus deletions/insertions in the flanking region.
* The gain in PD (loss in PM) from length-based to sequence-based data is particularly beneficial for mixture deconvolution [Novroski et al., 2016; Devesse et al, 2018].
* The increase in TPI through sequence-based data will be of great help in kinship studies [Staadig et al, 2019].
Penta_D[CE2.2]-chr21-hg19 45056086-45056150 [AAAGA]5 45056081-45056093-delAAAAGAAAGAAAA
D19S433[CE13.2]-chr19-hg19 30417142-30417197 [CCTT]12 ccta CCTT cttt CCTT 30417137-3041713-delGA
* The Spanish data set is expected to:- provide allele frequencies of STR sequencing for forensic casework applications- support implementation of MPS technology in forensic laboratories
* It is important to highlight the need for more population studies to enrich the allele frequencies of sequence-based STR genotypes.
The Faculty
Institute of Legal Medicine, Medical University of Innsbruck. Austria
Institute of Legal Medicine and ForensicScience, Charité - Universitätsmedizin Berlin. Germany
Center for Human Identification at the University of North Texas Health Science Center. USA
National Institute of Toxicology and Forensic Sciences. Madrid Department. Spain
Follow us on https://www.researchgate.net/project/DNASEQEX
Thanks for your attention!!
This project has been funded with support from the European Commission (grantHOME/2014/ISFP/AG/LAWX/4000007135 under the Internal Security Funding Policeprogramme of the European Commission---Directorate General Justice and Home Affairs). Thispublication reflects the views only of the authors, and the European Commission cannot be heldresponsible for any use which may be made of the information contained therein.
Acknowledgements
Thermo Fisher Scientific Inc. Provider of EA Precision ID GlobalFiler® PanelsPrototypes, Precision ID GlobalFiler® NGS STR Panel v2 and Converge v2.1 software.The authors would like to thank Matt Phipps for technical support.
The authors would like to thank members of LIMS Administrators Team of the General Subdirectorate ofNew Technologies of Justice (SGNTJ) of the Ministry of Justice (Spain) for their helpful technical support.
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