Introduction of the Python script STRinNGS for analysis of STR regions in FASTQ or BAM files and expansion of the Danish STR sequence database to 11 STRs.

This work introduces the in-house developed Python application STRinNGS for analysis of STR sequence elements in BAM or FASTQ files. STRinNGS identifies sequence reads with STR loci by their flanking sequences, it analyses the STR sequence and the flanking regions, and generates a report with the assigned SNP-STR alleles. The main output file from STRinNGS contains all sequences with read counts above 1% of the total number of reads per locus. STR sequences are automatically named according to the nomenclature used previously and according to the repeat unit definitions in STRBase (http://www.cstl.nist.gov/strbase/). The sequences are named with (1) the locus name, (2) the length of the repeat region divided by the length of the repeat unit, (3) the sequence(s) of the repeat unit(s) followed by the number of repeats and (4) variations in the flanking regions. Lower case letters in the main output file are used to flag sequences with previously unknown variations in the STRs. SNPs in the flanking regions are named by their "rs" numbers and the nucleotides in the SNP position. Data from 207 Danes sequenced with the Ion Torrent™ HID STR 10-plex that amplified nine STRs (CSF1PO, D3S1358, D5S818, D7S820, D8S1179, D16S539, TH01, TPOX, vWA), and Amelogenin was analysed with STRinNGS. Sequencing uncovered five common SNPs near four STRs and revealed 20 new alleles in the 207 Danes. Three short homopolymers in the D8S1179 flanking regions caused frequent sequencing errors. In 29 of 3726 allele calls (0.8%), sequences with homopolymer errors were falsely assigned as true alleles. An in-house developed script in R compensated for these errors by compiling sequence reads that had identical STR sequences and identical nucleotides in the five common SNPs. In the output file from the R script, all SNP-STR haplotype calls were correct. The 207 samples and six additional samples were sequenced for D3S1358, D12S391, and D21S11 using the 454 GS Junior platform in this and a previous work. Overall, next generation sequencing (NGS) of the 11 STRs lowered the mean match probability 386 times and increased the typical paternity indexes (i.e. the geometric mean) for trios and duos 47 and 23 times, respectively, compared to the traditional PCR-CE typing of the same population.

Second generation sequencing of three STRs D3S1358, D12S391 and D21S11 in Danes and a new nomenclature for sequenced STR alleles.

Second generation sequencing (SGS) may revolutionize the field of forensic STR typing. Two of the essential requirements for implementation of an SGS based approach for forensic investigations are (1) establishment of adequate frequency databases and (2) adoption of a new STR nomenclature. We report the STR sequences and allele frequencies of three STR loci: D3S1358, D12S391 and D21S11 in 197 unrelated Danes. We used a new STR nomenclature that depicts the locus name used in forensic genetics, the length of the repeat region divided by the repeat length (typically 4 nucleotides) and detailed sequence information of possible sub-repeats and SNPs within the amplified fragment.

Characterization of mutations and sequence variants in the D21S11 locus by next generation sequencing.

We sequenced the D21S11 locus in 77 individuals from Danish paternity cases using 454 FLX next generation sequencing (NGS) technology. All samples were also typed with the AmpFlSTR Profiler Plus or the AmpFlSTR Identifiler PCR Amplification kits as part of paternity investigations. In 18 of the confirmed trios, a genetic inconsistency was observed between one of the parents and the child at the D21S11 locus. NGS of the D21S11 locus revealed which allele had mutated from which parent to the child in 13 of these trios. All characterized mutations could be explained by single-step mutations in the longest sub-repeat of D21S11. A total of 53 of the 77 sequenced samples originated from unrelated individuals. Twenty different D21S11 alleles were detected by NGS in these individuals whereas only 13 different alleles were observed with fragment analysis. Several alleles had the same lengths but different sequences, e.g. four and three different alleles were detected by NGS with lengths determined by CE corresponding to allele 30 and allele 31, respectively.

Forensic and phylogeographic characterisation of mtDNA lineages from Somalia.

The African mitochondrial (mt) phylogeny is coarsely resolved but the majority of population data generated so far is limited to the analysis of the first hypervariable segment (HVS-1) of the control region (CR). Therefore, this study aimed on the investigation of the entire CR of 190 unrelated Somali individuals to enrich the severely underrepresented African mtDNA pool. The majority (60.5 %) of the haplotypes were of sub-Saharan origin with L0a1d, L2a1h and L3f being the most frequently observed haplogroups. This is in sharp contrast to previous data reported from the Y-chromosome, where only about 5 % of the observed haplogroups were of sub-Saharan provenance. We compared the genetic distances based on population pairwise F (st) values between 11 published East, Central and North African as well as western Asian populations and the Somali sequences and displayed them in a multi-dimensional scaling plot. Genetic proximity evidenced by clustering roughly reflected the relative geographic location of the populations. The sequences will be included in the EMPOP database ( www.empop.org ) under accession number EMP00397 upon publication (Parson and Dür Forensic Sci Int Genet 1:88-92, 2007).

Typing of 30 insertion/deletions in Danes using the first commercial indel kit--Mentype® DIPplex.

In this study, we tested the first commercial kit with insertion/deletion (indel) polymorphisms, the Mentype(®) DIPplex PCR Amplification Kit (DIPplex kit). A total of 30 biallelic autosomal indels and Amelogenin were amplified with the DIPplex kit. All loci were amplified in one PCR multiplex and all amplicon lengths were shorter than 160 bp. Full indel profiles were generated from as little as 100 pg of DNA. A total of 117 individuals from Danish paternity cases were successfully typed. No deviation from Hardy-Weinberg equilibrium was observed for any of the indels. The combined mean match probability was 3.3 × 10(-13), the mean paternity exclusion probability was 99.7% and the typical paternity indices for trios and duos were 2350 and 165, respectively. Furthermore, we typed five highly degraded DNA samples with the DIPplex kit, the AmpFlSTR(®) SGM Plus kit and the AmpFlSTR(®) SEfiler Plus kit. Full indel profiles were obtained with the DIPplex kit, whereas only partial profiles were obtained with the STR kits. In general, the DIPplex kit performed well and it would be a valuable assay for forensic genetic testing, especially in crime cases with partially degraded DNA or low amounts of template DNA. However, some difficulties with pull-ups were observed at DNA concentrations of 1000 pg. Rearrangement of the allele windows by changing the lengths of some of the PCR primers would greatly improve the assay, and more robustness towards higher amounts of DNA would allow the use of the DIPplex kit without prior quantification of the samples.

High-throughput sequencing of core STR loci for forensic genetic investigations using the Roche Genome Sequencer FLX platform.

The analysis and profiling of short tandem repeat (STR) loci is routinely used in forensic genetics. Current methods to investigate STR loci, including PCR-based standard fragment analyses and capillary electrophoresis, only provide amplicon lengths that are used to estimate the number of STR repeat units. These methods do not allow for the full resolution of STR base composition that sequencing approaches could provide. Here we present an STR profiling method based on the use of the Roche Genome Sequencer (GS) FLX to simultaneously sequence multiple core STR loci. Using this method in combination with a bioinformatic tool designed specifically to analyze sequence lengths and frequencies, we found that GS FLX STR sequence data are comparable to conventional capillary electrophoresis-based STR typing. Furthermore, we found DNA base substitutions and repeat sequence variations that would not have been identified using conventional STR typing.

Typing of 49 autosomal SNPs by SNaPshot in the Slovenian population.

A total of 157 unrelated individuals residing in Slovenia were typed for 49 of the autosomal single nucleotide polymorphisms (SNPs) in the SNPforID 52plex with the SNaPshot assay. We obtained full SNP profiles in all but one individual and perfect concordance was obtained in duplicated analyses. Allele frequencies are presented for the 49 SNPs. No deviation from HWE was observed for any SNP. F(IS) and F(ST) were estimated. A principal coordinate analysis performed on six populations (Slovenian, Danish, Somali, Greenland, Turkish and Chinese) showed that the Slovenian population grouped with the Danish population. The mean power of discrimination for the Slovenian population was 1.1 x 10(-19), and the mean exclusion probability for trios was 99.96%.

Validation of a single nucleotide polymorphism (SNP) typing assay with 49 SNPs for forensic genetic testing in a laboratory accredited according to the ISO 17025 standard.

A multiplex assay with 49 autosomal single nucleotide polymorphisms (SNPs) developed for human identification was validated for forensic genetic casework and accredited according to the ISO 17025 standard. The multiplex assay was based on the SNPforID 52plex SNP assay [J.J. Sanchez, C. Phillips, C. Børsting, K. Balogh, M. Bogus, M. Fondevila, C.D. Harrison, E. Musgrave-Brown, A. Salas, D. Syndercombe-Court, P.M. Schneider, A. Carracedo, N. Morling, A multiplex assay with 52 single nucleotide polymorphisms for human identification, Electrophoresis 27 (2006) 1713-1724], where 52 fragments were amplified in one PCR reaction. The SNPs were analysed by single base extension (SBE) and capillary electrophoresis. Twenty-three of the original SBE primers were altered to improve the overall robustness of the assay and to simplify the analysis of the SBE results. A total of 216 samples from 50 paternity cases and 33 twin cases were typed at least twice for the 49 SNPs. All electropherograms were analysed independently by two expert analysts prior to approval. Based on these results, detailed guidelines for analysis of the SBE products were developed. With these guidelines, the peak height ratio of a heterozygous allele call or the signal to noise ratio of a homozygous allele call is compared with previously obtained ratios. A laboratory protocol for analysis of SBE products was developed where allele calls with unusual ratios were highlighted to facilitate the analysis of difficult allele calls. The guidelines for allele calling proved to be highly efficient for the detection of DNA mixtures and contaminated DNA preparations. DNA from two individuals was mixed in seven different ratios ranging from 1:1 to 1:10; all mixtures were easily identified as mixtures.

Linkage disequilibrium mapping of a breast cancer susceptibility locus near RAI/PPP1R13L/iASPP.

BACKGROUND: Previous results have suggested an association of the region of 19q13.3 with several forms of cancer. In the present study, we investigated 27 public markers within a previously identified 69 kb stretch of chromosome 19q for association with breast cancer by using linkage disequilibrium mapping. The study groups included 434 postmenopausal breast cancer cases and an identical number of individually matched controls. METHODS AND RESULTS: Studying one marker at a time, we found a region spanning the gene RAI (alias PPP1R13L or iASPP) and the 5' portion of XPD to be associated with this cancer. The region corresponds to a haplotype block, in which there seems to be very limited recombination in the Danish population. Studying combinations of markers, we found that two to four neighboring markers gave the most consistent and strongest result. The haplotypes with strongest association with cancers were located in the gene RAI and just 3' to the gene. Coinciding peaks were seen in the region of RAI in groups of women of different age. In a follow-up to these results we sequenced 10 cases and 10 controls in a 44 kb region spanning the peaks of association. This revealed 106 polymorphisms, many of which were not in the public databases. We tested an additional 44 of these for association with disease and found a new tandem repeat marker, called RAI-3'd1, located downstream of the transcribed region of RAI, which was more strongly associated with breast cancer than any other marker we have tested (RR = 2.44 (1.41-4.23, p = 0.0008, all cases; RR = 6.29 (1.49-26.6), p = 0.01, cases up to 55 years of age). CONCLUSION: We expect the marker RAI-3'd1 to be (part of) the cause for the association of the chromosome 19q13.3 region's association with cancer.

SNP genotyping using microsphere-linked PNA and flow cytometric detection.

BACKGROUND: Single nucleotide polymorphisms (SNPs) represent the most frequent form of genetic variations. Some of the most sensitive methods for SNP genotyping employ synthetic oligonucleotides, such as the peptide nucleic acid (PNA). We introduce a new method combining allele-specific hybridization, PNA technology, and flow cytometric detection. We tested the design by genotyping a Danish basal cell carcinoma cohort of 80 individuals for an A/C SNP in exon 6 of the XPD gene. METHODS: Genomic DNA was amplified by a two-step polymerase chain reaction (PCR) in the presence of fluorescein-dyed primers and fluorescein-12-dUTP. The allele-specific PNA molecules were covalently coupled to carboxylated microspheres with and without rhodamine. Allele-specific hybridization between PCR products and immobilized PNA was carried out at 60 degrees C followed by flow cytometric detection. RESULTS: We present a fully functional two-bead genotyping system based on PNA capture and flow cytometric detection used for the correct and fast regenotyping of a Danish basal cell carcinoma cohort. CONCLUSIONS: This new assay presents a simple, rapid, and robust method for SNP genotyping for laboratories equipped with a standard flow cytometer. Moreover, this system offers potential for multiplexing and will be operational for middle-scale genotyping.

Short PNA molecular beacons for real-time PCR allelic discrimination of single nucleotide polymorphisms.

The typing of a single nucleotide polymorphism with DNA probes is sometimes problematic because of the limited discriminating power of long DNA probes. As an alternative to existing assays, we have developed a real-time PCR assay for the genotyping of single nucleotide polymorphisms using short peptide nucleic acid (PNA) molecular beacons. A single nucleotide polymorphism in exon 6 of the XPD gene was chosen as the model system. The genotyping experiments were performed in the ABI 7700 using beacons labeled with either fluorescein or JOE, and in the Lightcycler using a fluorescein labeled beacon. QSY-7 was used as the quencher in all the beacons. The result of the genotyping was the same on both instruments and was in agreement with a previously performed RFLP genotyping of 79 samples. The length of PNA molecular beacons is significantly shorter than that of TaqMan or Lightcycler probes, making probe design and genotype discrimination easier.

Multiple single nucleotide polymorphisms on human chromosome 19q13.2-3 associate with risk of Basal cell carcinoma.

In this paper, we present evidence that alleles of several polymorphisms in the chromosomal region 19q13.2-3, encompassing the genes RAI and XPD, are associated with occurrence of basal cell carcinoma in Caucasian Americans. The association of one of these, RAI-intron1, is sufficiently strong to make mass significance unlikely (P = 0.004, chi(2)). We interpret our combined data to indicate that a specific haplotype partly defined by the alleles of three single nucleotide polymorphisms, RAI intron1(G), RAI exon6(T), and XPD exon 6(C), is associated with a protective gene variant in a region spanning from XPD to ERCC1.

Association of chromosome 19q13.2-3 haplotypes with basal cell carcinoma: tentative delineation of an involved region using data for single nucleotide polymorphisms in two cohorts.

We have previously used single nucleotide polymorphisms to detect an association of basal cell carcinoma (BCC) in Caucasian Americans and Danes with the genome region 19q13.2-3, which contains several genes involved in the nucleotide excision repair of DNA. In this exploratory paper we have extended the data and used them in a chromosomal scan. The results indicate the presence of a gene variation modulating the risk of developing BSS in a submegabase region including and surrounding the gene RAI. Specifically, persons that are homozygous for the haplotype RAI intron 1(A) RAI exon 6(A) appear at increased risk for BCC. In addition, we have looked for possible synergisms between all pairs of markers. We find that a marker in GLTSCR1, presumably separated from RAI by several million bases, supplements the most significant marker in RAI in separating cases from controls, which may suggest the presence of an independent, risk-modulating variation in this second gene region.