Alignment and annotation of forensic STR data generated from MPS analysis: Notes from the sequence workbench

Transcription

1 Alignment and annotation of forensic STR data generated from MPS analysis: Notes from the sequence workbench Chris Phillips, Katherine Gettings, Jonathan King, Christophe van Neste, Walther Parson

2 Capillary electrophoresis to sequencing of forensic STRs D12S391: X.3 intermediate

3 Capillary electrophoresis to sequencing of forensic STRs D12S391: [AGAT]a [AGAC]b [AGAT]c

4 Sanger to MPS

5 Sanger to MPS Alleles

6 Sanger to MPS A 19 B C 20 D 91 Alleles E F G H 23 I J

7 STR sequence allele nomenclature guidelines

8 STR sequence allele nomenclature guidelines Outlined 8 practical considerations for analysis of forensic STR data generated by MPS Stated analysis, export and storage of MPS data should be as full sequence strings (as in EMPOP) Recommended sequence alignments use the most up-to-date human genome assembly Suggested a framework for variant annotation with a curated sequence template file (S1) The published sequence template file S1 will be transferred to an FTP site with a change-log MPS STR allele nomenclature needs careful discussions and planning - a broader based project Genomic descriptions of novel STRs require systematic checks too: D5S2500 HDplex AGCU

9 STR sequence allele nomenclature guidelines Outlined 8 practical considerations for analysis of forensic STR data generated by MPS Stated analysis, export and storage of MPS data should be as full sequence strings (as in EMPOP) Recommended sequence alignments use the most up-to-date human genome assembly Suggested a framework for variant annotation with a curated sequence template file (S1) The published sequence template file S1 will be transferred to an FTP site with a change-log MPS STR allele nomenclature needs careful discussions and planning - a broader based project Genomic descriptions of novel STRs require systematic checks too: D5S2500 HDplex AGCU

10 STR sequence allele nomenclature guidelines Outlined 8 practical considerations for analysis of forensic STR data generated by MPS Stated analysis, export and storage of MPS data should be as full sequence strings (as in EMPOP) Recommended sequence alignments use the most up-to-date human genome assembly Suggested a framework for variant annotation with a curated sequence template file (S1) The published sequence template file S1 will be transferred to an FTP site with a change-log MPS STR allele nomenclature needs careful discussions and planning - a broader based project Genomic descriptions of novel STRs require systematic checks too: D5S2500 HDplex AGCU

11 STR sequence allele nomenclature guidelines Outlined 8 practical considerations for analysis of forensic STR data generated by MPS Stated analysis, export and storage of MPS data should be as full sequence strings (as in EMPOP) Recommended sequence alignments use the most up-to-date human genome assembly Suggested a framework for variant annotation with a curated sequence template file (S1) The published sequence template file S1 will be transferred to an FTP site with a change-log MPS STR allele nomenclature needs careful discussions and planning - a broader based project Genomic descriptions of novel STRs require systematic checks too: D5S2500 HDplex AGCU

12 TPOX The repeat motif for each STR marker is listed according to the International Society of Forensic Genetics (ISFG) recommendation that the repeat sequence motif be defined so that the first 50- nucleotides on the GenBank forward strand define the repeat motif used (56) [AATG]a

13 TPOX The repeat motif for each STR marker is listed according to the International Society of Forensic Genetics (ISFG) recommendation that the repeat sequence motif be defined so that the first 50- nucleotides on the GenBank forward strand define the repeat motif used (56) [AATG]a 5 3 rs

14 TPOX The repeat motif for each STR marker is listed according to the International Society of Forensic Genetics (ISFG) recommendation that the repeat sequence motif be defined so that the first 50- nucleotides on the GenBank forward strand define the repeat motif used (56) [AATG]a 5 3 rs Data Slicer Chr Position Ref allele Alt allele Data Slicer

15 STR sequence allele nomenclature guidelines Outlined 8 practical considerations for analysis of forensic STR data generated by MPS Stated analysis, export and storage of MPS data should be as full sequence strings (as in EMPOP) Recommended sequence alignments use the most up-to-date human genome assembly Suggested a framework for variant annotation with a curated sequence template file (S1) The published sequence template file S1 will be transferred to an FTP site with a change-log MPS STR allele nomenclature needs careful discussions and planning - a broader based project Genomic descriptions of novel STRs require systematic checks too: D5S2500 HDplex AGCU

16 STR sequence allele nomenclature guidelines Outlined 8 practical considerations for analysis of forensic STR data generated by MPS Stated analysis, export and storage of MPS data should be as full sequence strings (as in EMPOP) Recommended sequence alignments use the most up-to-date human genome assembly Suggested a framework for variant annotation with a curated sequence template file (S1) The published sequence template file S1 will be transferred to an FTP site with a change-log MPS STR allele nomenclature needs careful discussions and planning - a broader based project Genomic descriptions of novel STRs require systematic checks too: D5S2500 HDplex AGCU oversight group

17 STR sequence allele nomenclature guidelines Outlined 8 practical considerations for analysis of forensic STR data generated by MPS Stated analysis, export and storage of MPS data should be as full sequence strings (as in EMPOP) Recommended sequence alignments use the most up-to-date human genome assembly Suggested a framework for variant annotation with a curated sequence template file (S1) The published sequence template file S1 will be transferred to an FTP site with a change-log MPS STR allele nomenclature needs careful discussions and planning - a broader based project Genomic descriptions of novel STRs require systematic checks too: D5S2500 HDplex AGCU oversight group

18 STR sequence allele nomenclature guidelines Outlined 8 practical considerations for analysis of forensic STR data generated by MPS Stated analysis, export and storage of MPS data should be as full sequence strings (as in EMPOP) Recommended sequence alignments use the most up-to-date human genome assembly Suggested a framework for variant annotation with a curated sequence template file (S1) The published sequence template file S1 will be transferred to an FTP site with a change-log MPS STR allele nomenclature needs careful discussions and planning - a broader based project Genomic descriptions of novel STRs require systematic checks too: D5S2500 HDplex AGCU oversight group

19 MPS analysis of STRs with the CEPH diversity panel Sequenced 27 A-STRs, 24 Y-STRs and 7 X- STRs in the CEPH panel Checked genotyping concordance with CE analysis of the same 944 samples made in 2010 Transferred the reported STR sequences to Excel and aligned under the current human reference sequence at each locus Used simple macros to reverse-compliment when needed and order sequences alphabetically Cross-checked flanking SNPs plus RR and flanking Indels with 1000 Genomes P-III data

20 20 CODIS-A Autosomal STRs Sized alleles from CE CEPH-wide average Heterozygosity of 81.2% D1S1656 D2S1338 D12S391 FGA D18S51 D21S11 D19S433 Sequenced alleles from MPS No sequence variation detected so far D21S11 FGA TH01 VWA D8S1179 D13S317 D7S820 TH01 D16S539 D10S1248 D2S441 D5S818 D22S1045 D3S1358 CSF1PO!0.15!0.1! % -10% -5% 5% 10% 15% TPOX

21 20 CODIS-A Autosomal STRs Sized alleles from CE CEPH-wide average Heterozygosity of 81.2% D1S1656 D2S1338 D12S391 FGA D18S51 D21S11 D19S433 Sequence reads Sequenced alleles from MPS No sequence variation detected so far D21S11 FGA TH01 VWA D8S1179 D13S317 D7S820 TH01 Allele D16S539 D10S1248 D2S441 D5S818 D22S1045 D3S1358 CSF1PO!0.15!0.1! % -10% -5% 5% 10% 15% TPOX

22 Alignment: human genome assemblies follow a set system short p arm 5 The forward strand starts at the first 5 p-arm nucleotide - to the last 3 q-arm nucleotide of each chromosome long q arm 3 CRS rcrs

23 Alignment: human genome assemblies follow a set system short p arm 5 The forward strand starts at the first 5 p-arm nucleotide - to the last 3 q-arm nucleotide of each chromosome long q arm 3 CRS rcrs 14: coordinates map to unique positions

24 Two main current human genome assemblies 5 1:1 GRCh38 (hg38) December 2013 short p arm long q arm 3 CRS rcrs 22:

25 Two main current human genome assemblies 5 1:1 GRCh38 (hg38) December 2013 GRCh37 (hg19) February CRS rcrs 22: :

26 Frequency of new assembly builds 5 GRCh38 (hg38) December 2013 GRCh37 (hg19) February 2009 NCBI 36 (hg18) March 2006 NCBI 35 (hg17) May 2004 NCBI 34 (hg16) July CRS rcrs

27 Ten A-STR repeat descriptions use reverse strand direction D2S1338 [TGCC]a [TTCC]b - STRbase D2S1338 [GGAA]a [GGCA]b - reference genome Coding strand W. Bär, B. Brinkmann, B. Budowle, A. Carracedo, P. Gill, P. Lincoln, DNA recommendations. Further report of the DNA Commission of the ISFH regarding the use of short tandem repeat systems. International Society for Forensic Haemogenetics, Int. J. Legal Med. 110 (1997)

28 Both double Y-STR loci are tandem inversions DYS385 a/b a=[gaaa]p b=[gaaa]q a=[tttc]p b=[gaaa]q [AAAG]a [GTAG]b [GAAG]c [AAAG]d GAAG [AAAG]e [GAAG]f [AAAG]g DYF387S [CTTT]a [CTTC]b [CTTT]c [CTTC]d [CTTT]e CTTC [CTAC]f [CTTT]g (fragment 2 description changes)

29 Both double Y-STR loci are tandem inversions DYS385 a/b a=[gaaa]p b=[gaaa]q a=[gaaa]p b=[tttc]q b a [AAAG]a [GTAG]b [GAAG]c [AAAG]d GAAG [AAAG]e [GAAG]f [AAAG]g DYF387S [CTTT]a [CTTC]b [CTTT]c [CTTC]d [CTTT]e CTTC [CTAC]f [CTTT]g (fragment 2 description changes)

30 GRCh37 and 38 match in 55 of 58 STRs compared so far DYS437, DYS438, DYS439 show sequence differences in their repeat regions between GRCh37 and GRCh Genomes still uses GRCh37 co-ordinates for all sequence and variant data

31 20 CODIS-A Autosomal STRs Sized alleles from CE CEPH-wide average Heterozygosity of 81.2% D1S1656 D2S1338 D12S391 FGA D18S51 D21S11 D19S433 Sequenced alleles from MPS No sequence variation detected so far D21S11 FGA TH01 VWA D8S1179 D13S317 D7S820 TH01 D16S539 D10S1248 D2S441 D5S818 D22S1045 D3S1358 CSF1PO!0.15!0.1! % -10% -5% 5% 10% 15% TPOX

32 D18S51 Reported Sequence (repeat sequence +10 nt) 5 3

33 D18S51 Reported Sequence (repeat sequence +10 nt) 3

34 D18S51 Repeat sequence 10 nt 3

35 D18S51 Repeat sequence 10 nt 3

36 D13S317 Reported Sequence (repeat sequence + 31 nt) 5 3

37 D13S317 Repeat sequence 31 nt

38 D19S433 Reported Sequence (18 nt + repeat sequence = 48 / 16 nt) 5 3

39 D19S433 Reported Sequence (18 nt + repeat sequence = 48 / 16 nt) 5 3

40 D19S433 Reported Sequence (18 nt + repeat sequence = 48 / 16 nt) 5 3

41 D19S nt Repeat sequence of: 48 nt and: 16 nt 5 3

42 D19S nt Repeat sequence of: 48 nt and: 16 nt 5 3

43 How to deal with less or more nucleotides in the analysed repeat region compared to the reference sequence? _

44 How to deal with less or more nucleotides in the analysed repeat region compared to the reference sequence? _

45 Towards a lean form of STR annotation

46 Towards a lean form of STR annotation e.g. D13S A C T G - + A C T G

47 Towards a lean form of STR annotation e.g. D13S A C T G - + A C T G RR start RR stop Anchor nt

48 Towards a lean form of STR annotation e.g. D13S features in a grid of 2,457 (each with 13:x coordinates) = 8%

49 D21S11 is the most challenging STR to describe 23 size alleles > 92 sequence alleles TA TCCATA [TCTA]a [TCTG]b [TCTA]c TA [TCTA]d TCA [TCTA]e TCCATA [TCTA]f 126 nt of reference sequence > 213 nt of all observed positions

50 D21S11 is the most challenging STR to describe RR start 23 size alleles > 92 sequence alleles (4-fold rise in variation) RR stop TA TCCATA [TCTA]a [TCTG]b [TCTA]c TA [TCTA]d TCA [TCTA]e TCCATA [TCTA]f 126 nt of reference sequence > 213 nt of all observed positions ad hoc 11-nt insertion in AFR singleton [TATCTA] [TCTA]N motif not in reference sequence

51 D21S11 is the most challenging STR to describe RR start 23 size alleles > 92 sequence alleles (4-fold rise in variation) RR stop TA TCCATA [TCTA]a [TCTG]b [TCTA]c TA [TCTA]d TCA [TCTA]e TCCATA [TCTA]f [TATCTA] [TCTA]g 126 nt of reference sequence > 213 nt of all observed positions +10 novel nt ad hoc 11-nt insertion in AFR singleton [TATCTA] [TCTA]N motif not in reference sequence

52 Concluding remarks Obtaining the whole sequence string allows more complete annotation of complex Indel and nucleotide re-arrangements at the repeat region as well as the compilation of flanking region variants from a forensic MPS analysis A standard, unified framework of sequence alignment based on the human genome reference assembly will make it easier to regulate and evolve a sequence-based STR allele nomenclature system Definition of the repeat region start and stop nucleotides in the reference sequence is the key step to ensuring backwards compatibility of existing size-allele nomenclature to the next step of assigning names to sequences To facilitate the adoption of standardised alignment and annotation practices for labs collecting population data with MPS, an Excel sequence template file will be published as an FTP file with regular scrutiny, updates and full change-log details. Since publication made 16 annotation changes.

53 Concluding remarks Obtaining the whole sequence string allows more complete annotation of complex Indel and nucleotide re-arrangements at the repeat region as well as the compilation of flanking region variants from a forensic MPS analysis A standard, unified framework of sequence alignment based on the human genome reference assembly will make it easier to regulate and evolve a sequence-based STR allele nomenclature system Definition of the repeat region start and stop nucleotides in the reference sequence is the key step to ensuring backwards compatibility of existing size-allele nomenclature to the next step of assigning names to sequences To facilitate the adoption of standardised alignment and annotation practices for labs collecting population data with MPS, an Excel sequence template file will be published as an FTP file with regular scrutiny, updates and full change-log details. Since publication made 16 annotation changes.

54 Concluding remarks Obtaining the whole sequence string allows more complete annotation of complex Indel and nucleotide re-arrangements at the repeat region as well as the compilation of flanking region variants from a forensic MPS analysis A standard, unified framework of sequence alignment based on the human genome reference assembly will make it easier to regulate and evolve a sequence-based STR allele nomenclature system Definition of the repeat region start and stop nucleotides in the reference sequence is the key step to ensuring backwards compatibility of existing size-allele nomenclature to the next step of assigning names to sequences To facilitate the adoption of standardised alignment and annotation practices for labs collecting population data with MPS, an Excel sequence template file will be published as an FTP file with regular scrutiny, updates and full change-log details. Since publication made 16 annotation changes.

55 Concluding remarks Obtaining the whole sequence string allows more complete annotation of complex Indel and nucleotide re-arrangements at the repeat region as well as the compilation of flanking region variants from a forensic MPS analysis A standard, unified framework of sequence alignment based on the human genome reference assembly will make it easier to regulate and evolve a sequence-based STR allele nomenclature system Definition of the repeat region start and stop nucleotides in the reference sequence is the key step to ensuring backwards compatibility of existing size-allele nomenclature to the next step of assigning names to sequences To facilitate the adoption of standardised alignment and annotation practices for labs collecting population data with MPS, an Excel sequence template file will be published as an FTP file with regular scrutiny, updates and full change-log details. Since publication made 16 annotation changes.

56 It is important that guidelines for any complex task are precise and easy to follow

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