Highly effective DNA extraction method for nuclear short tandem repeat testing of skeletal remains from mass graves

Aim

To quantitatively compare a silica extraction method with a commonly used phenol/chloroform extraction method for DNA analysis of specimens exhumed from mass graves.

Methods

DNA was extracted from twenty randomly chosen femur samples, using the International Commission on Missing Persons (ICMP) silica method, based on Qiagen Blood Maxi Kit, and compared with the DNA extracted by the standard phenol/chloroform-based method. The efficacy of extraction methods was compared by real time polymerase chain reaction (PCR) to measure DNA quantity and the presence of inhibitors and by amplification with the PowerPlex 16 (PP16) multiplex nuclear short tandem repeat (STR) kit.

Results

DNA quantification results showed that the silica-based method extracted on average 1.94 ng of DNA per gram of bone (range 0.25-9.58 ng/g), compared with only 0.68 ng/g by the organic method extracted (range 0.0016-4.4880 ng/g). Inhibition tests showed that there were on average significantly lower levels of PCR inhibitors in DNA isolated by the organic method. When amplified with PP16, all samples extracted by silica-based method produced 16 full loci profiles, while only 75% of the DNA extracts obtained by organic technique amplified 16 loci profiles.

Conclusions

The silica-based extraction method showed better results in nuclear STR typing from degraded bone samples than a commonly used phenol/chloroform method.Analysis by nuclear short tandem repeats (STR) has been proven invaluable for identifications in mass fatality incidents such as plane crashes, terrorist attacks, natural disasters, armed conflict, or any other case where traditional methods of identification are insufficient (1-4). DNA-STR testing often provides the strongest evidence of identity in cases of high degradation of human remains.Bone and teeth are excellent sources of DNA for human identity testing which uses STRs or mitochondrial DNA (mtDNA) (5-7), and in cases of extreme degradation may be the only suitable material available for successful typing. However, relatively specialized techniques are required for the extraction of DNA from bone, particularly when the bones have been exposed to adverse environmental conditions and DNA is degraded and/or present in low amounts. The physical and chemical barriers in bone that protect the DNA from environment and microbial assault also hinder the access of reagents in the extraction process (8,9). Another major difficulty is co-extraction of compounds inhibitory to the polymerase chain reaction (PCR) (10-13), particularly with skeletal samples exposed to soil or other environmental contaminants.It is beyond the scope of this article to review the wide variety of DNA extraction methods that have been reported for bone, but two of the major approaches, with innumerable variations, are 1) organic extraction methods involving phenol/chloroform (7,14,15) and 2) silica-binding methods where guanidinium-based chaotropic salts are used both to disrupt proteins, as well as mediate highly specific binding of DNA to silica particles via ionic salt bridges (12,16,17). Because of the high specificity of DNA binding to silica, inhibition is often less of a problem with this method, and silica binding purifications are sometimes used as secondary clean-up steps after organic extractions to remove inhibitors (15).Nuclear STR profiling has been found highly successful by the International Commission on Missing Persons (ICMP) in their large-scale effort to identify skeletal remains from mass graves in the former Yugoslavia (5,6). In this work, the ICMP has used a silica-binding method based on substantial protocol modifications of a commercially available DNA extraction kit. This DNA extraction method was also used very successfully on more than 1800 bone and tooth samples from the 2004 Asian tsunami that were processed by the ICMP (our unpublished data).In this article, we compared the quantitative performance of the ICMP-developed silica extraction method with a standard organic phenol/chloroform extraction method on the same set of femur samples. Also, we evaluated the levels of PCR inhibitors present in the extract, as assessed by quantitative PCR with internal controls and the amplification obtained with multiplex nuclear STR testing. This comparative study will hopefully assist other groups in adopting simple and effective protocols for DNA isolation from degraded bone in casework relating to mass disasters, terrorist attacks, or mass graves.     

基于测序的人类白细胞抗原分型和PCR短串联重复序列技术在人胚胎干细胞鉴定中的应用

目的探讨基于测序的人类白细胞抗原分型(HLA-sequencing-based typing,HLA-SBT)和PCR短串联重复序列(short tandem repeat,STR)技术在人胚胎干细胞(human embryonic stem cell,hESC)应用前检测中的运用,建立人胚胎干细胞系的基因型档案。方法人胚胎干细胞系SYSU-1、SYSU-3,分别培养到20代、40代,应用PCR寡核苷酸特异测序探针(sequence specific olignucleotideprobe,SSO)技术检测两株细胞系的HLA-A、-B、-DR位点的低分辨分型,再利用HLA-SBT技术检测两株细胞系的HLA-A、-B、-DR位点的高分辨分型。应用PCR-STR技术检测两株细胞系的基因遗传标记。结果获得两株hESC细胞系的HLA高分辨分型和STR基因型。结论可以运用HLA-SBT和PCR-STR技术建立人胚胎干细胞应用前的基因型档案。     

基于测序的人类白细胞抗原分型和PCR短串联重复序列技术在人胚胎干细胞鉴定中的应用

目的 探讨基于测序的人类白细胞抗原分型(HLA-sequencing-based typing,HLA-SBT)和PCR短串联重复序列(short tandem repeat,STR)技术在人胚胎干细胞(human embryonic stem cell,hESC)应用前检测中的运用,建立人胚胎干细胞系的基因型档案.方法 人胚胎干细胞系SYSU-I、SYSU-3,分别培养到20代、40代,应用PCR寡核苷酸特异测序探针(sequence specific olignucleotide probe,SSO)技术检测两株细胞系的HLA-A、-B、-DR位点的低分辨分型,再利用HLA-SBT技术检测两株细胞系的HLA-A、-B、-DR位点的高分辨分型.应用PCR-STR技术检测两株细胞系的基因遗传标记.结果 获得两株hESC细胞系的HLA高分辨分型和STR基因型.结论 可以运用HLA-SBT和PCR-STR技术建立人胚胎干细胞应用前的基因型档案.     

Highly efficient nuclear DNA typing of the World War II skeletal remains using three new autosomal short tandem repeat amplification kits with the extended European Standard Set of loci

Aim

To perform an efficiency study of three new amplification kits with the extended European Standard Set (ESS) of loci for autosomal short tandem repeat (STR) typing of skeletal remains excavated from the World War II mass graves in Slovenia.

Methods

In the beginning of the 2011, we analyzed 102 bones and teeth using the PowerPlex ESX 17 System (Promega), AmpFiSTR NGM PCR Amplification Kit (Applied Biosystems), and Investigator ESSplex Kit (Qiagen). We cleaned the bones and teeth, removed surface contamination, and ground them into a powder using liquid nitrogen. Prior to DNA isolation with Biorobot EZ1 (Qiagen), 0.5 g bone or tooth powder was decalcified. Nuclear DNA of the samples was quantified using real-time polymerase chain reaction. All three kits used the same extract with the amplification conditions recommended by the manufacturers.

Results

We extracted up to 131 ng DNA/g of powder from the bones and teeth. All three amplification kits showed very similar efficiency, since DNA typing was successful with all amplification kits in 101 out of 102 bones and teeth, which represents a 99% success rate.

Conclusion

The commercially available ESX 17, ESSplex, and NGM kits are highly reliable for STR typing of World War II skeletal remains with the DNA extraction method optimized in our laboratory.DNA typing of bone and tooth samples has been successfully used in anthropological studies and forensic identification analysis (1,2). Nuclear DNA is the preferred genome of amplification for forensic purposes as it is individually specific and provides bi-parental kinship information (3). The success of DNA typing in old bones and teeth is often limited by small amounts of endogenous DNA, presence of polymerase chain reaction (PCR) inhibitors, DNA degradation, and an exceptional risk of contamination (4-6). Mitochondrial DNA testing has been regularly employed in the forensic identification of aged skeletal remains (7-10). Recently, some articles have reported a successful typing of nuclear short tandem repeats (STR) from ancient material using an increased number of cycles (11-18). In 2009 and 2010, new amplification kits were developed to meet the European Network of Forensic Institutes and the European DNA Profiling group recommendations for increasing the European Standard Set (ESS) of loci to improve its discrimination power and to fulfill the increasing requirements regarding sensitivity and reproducibility for the analysis of minute amounts of DNA by adopting five additional mini-STRs: D2S441, D10S1248, D22S1045, D1S1656, and D12S391 (19,20). Some validation, concordance, and population studies (21-28) have been published for new amplification kits with the extended ESS of loci. It was shown that the new kits are robust enough to genotype degraded DNA samples through the use of mini STR loci and have increased tolerance to common inhibitors and increased sensitivity to obtain full profiles from low-level DNA samples from casework (27,29,30). However, no study has been performed using new amplification kits on old skeletal remains. We attempted to obtain autosomal STR profiles from the World War II bones and teeth with three new commercially available amplification kits with the extended ESS of loci using the PCR protocols recommended by the manufacturers without increasing the number of cycles or any other modification of protocols.     

Multilocus variable-number tandem repeat typing of Mycobacterium ulcerans

The apparent genetic homogeneity of Mycobacterium ulcerans contributes to the poorly understood epidemiology of M. ulcerans infection. Here, we report the identification of variable number tandem repeat (VNTR) sequences as novel polymorphic elements in the genome of this species. A total of 19 potential VNTR loci identified in the closely related M. marinum genome sequence were screened in a collection of 23 M. ulcerans isolates, one Mycobacterium species referred to here as an intermediate species, and five M. marinum strains. Nine of the 19 loci were polymorphic in the three species (including the intermediate species) and revealed eight M. ulcerans and five M. marinum genotypes. The results from the VNTR analysis corroborated the genetic relationships of M. ulcerans isolates from various geographical origins, as defined by independent molecular markers. Although these results further highlight the extremely high clonal homogeneity within certain geographic regions, we report for the first time the discrimination of the two South American strains from Surinam and French Guyana. These findings support the potential of a VNTR-based genotyping method for strain discrimination within M. ulcerans and M. marinum.     

Variant alleles, triallelic patterns, and point mutations observed in nuclear short tandem repeat typing of populations in Bosnia and Serbia

Aim

To present a compendium of off-ladder alleles and other genotyping irregularities relating to rare/unexpected population genetic variation, observed in a large short tandem repeat (STR) database from Bosnia and Serbia.

Methods

DNA was extracted from blood stain cards relating to reference samples from a population of 32 800 individuals from Bosnia and Serbia, and typed using Promega’s PowerPlex®16 STR kit.

Results

There were 31 distinct off-ladder alleles were observed in 10 of the 15 STR loci amplified from the PowerPlex®16 STR kit. Of these 31 alleles, 3 have not been previously reported. Furthermore, 16 instances of triallelic patterns were observed in 9 of the 15 loci. Primer binding site mismatches that affected amplification were observed in two loci, D5S818 and D8S1179.

Conclusion

Instances of deviations from manufacturer’s allelic ladders should be expected and caution taken to properly designate the correct alleles in large DNA databases. Particular care should be taken in kinship matching or paternity cases as incorrect designation of any of these deviations from allelic ladders could lead to false exclusions.Commercial kits for forensic short tandem repeat (STR) multiplexes include allelic ladders that assist the user in allele designation by co-migrating with any commonly expected allelic variants in the population (1). In the selection of loci and other steps in the development of commercial STR kits, huge amounts of effort are put into screening population genetic variation to ensure that the locus varies in a simple and easy-to-interpret manner, and that allele frequencies are known for statistical interpretation. Allelic ladders are based on variation observed in the development process, but, inevitably, when large population samples are typed, variants are encountered that were not represented in the developmental screening. This is particularly true when the populations under testing differ from those that were studied during kit development.Practitioners of forensic STR typing need to be aware of the possibility of rare variants, so the variants can be recognized and dealt with properly in casework interpretation. If designated correctly, variant alleles can sometimes greatly increase the power of discrimination in DNA comparisons (2). In this regard, it is important that the forensic science community share information on the occurrence of these variants. In some contexts, these variants may actually occur rather commonly, and public documentation of their occurrence can save individual investigators much time and effort (3).Unexpected or “anomalous” genetic variation that can complicate STR typing takes a number of forms, with a variety of consequences on the testing results. New length variants not represented on the allelic ladder can be due to insertion/deletions of full repeat units, or to “microvariants” differing due to the insertion/deletion of single bases or partial repeats (2,4-6). In some cases, larger or smaller off-ladder alleles may fall within the allelic ladder of an adjacent locus (5,6), with the potential for significant confusion. Triallelic patterns (7-9) can be due to length mutations that occur and segregate during an individual’s development, or to localized duplication of a locus, or to chromosomal trisomy. Sequence variation, rather than length variation, can also have effects, particularly in the case of sequence differences in the amplification primer binding sites. Primer mismatches can result in complete amplification failure and cause null alleles, or can lower the peak height of affected alleles (10-18). Any of these anomalies can cause problems during interpretation of results if the analysts are not familiar with these occurrences.The International Commission on Missing Persons (ICMP) was founded in 1996 to address the issue of persons missing as a result of the conflicts that occurred during the breakup of the former Yugoslavia during the 1990s. DNA profiles are obtained from reference samples collected from living relatives which are entered into a DNA reference database (currently greater than 80 000 unique profiles). Likewise, DNA profiles generated from skeletal remains recovered from grave sites are entered into a missing persons DNA database (currently containing more than 12 000 unique profiles). Both databases are screened against each other on a daily basis, resulting in possible kinship matches.We report here a new compendium of all off-ladder variants/microvariants, primer binding site mutations, and instances of triallelic patterns that have been observed in a subset of the ICMP blood sample reference database representing ~ 32 800 individuals from Bosnia and Serbia, including Kosovo.     

Validation of a short tandem repeat multiplex typing system for genetic individualization of domestic cat samples

Aim

To conduct developmental validation studies on a polymerase chain reaction (PCR) based short tandem repeat (STR) multiplex typing system, developed for the purpose of genetic individualization and parentage testing in domestic cat samples.

Methods

To evaluate reproducibility of the typing system, the multiplex was amplified using DNA extracted from hair, blood, and buccal samples obtained from the same individual (n = 13). Additional studies were performed to evaluate the system’s species’ specificity, using 26 North American mammalian species and two prokaryotes Sacchromyces and Escherichia coli, sensitivity, and ability to identify DNA mixtures. Patterns of Mendelian inheritance and mutation rates for the 11 loci were directly examined in a large multi-generation domestic cat pedigree (n = 263).

Results

Our studies confirm that the multiplex system was species-specific for feline DNA and amplified robustly with as little as 125 picograms of genomic template DNA, demonstrating good product balance. The multiplex generated all components of a two DNA mixture when the minor component was one tenth of the major component at a threshold of 50 relative fluorescence units. The multiplex was reproducible in multiple tissue types of the same individual. Mutation rates for the 11 STR were within the range of sex averaged rates observed for Combined DNA Index System (CODIS) loci.

Conclusion

The cat STR multiplex typing system is a robust and reliable tool for the use of forensic DNA analysis of domestic cat samples.In the field of forensic DNA analysis, new methods and technology have revolutionized the analysis and detection of genetic variation for human identification (1-3). These advances have been extended to the analysis of DNA extracted from non-human specimens such as plants, bacteria, viruses, and domesticated animals (4). Genetic individualization of animal specimens has increasingly been included as key evidence in criminal investigations (5-11). It has been reported that pet hairs are invariably transferred to the clothing of those visiting the home of a pet owner (12). With approximately 73 million cats residing in one third of households in the United States (13), it is not surprising that cat hairs are often part of the physical evidence associated with crime scenes.Recently, a polymerase chain reaction (PCR) based short tandem repeat (STR) multiplex typing system has been developed for the use in genetic individualization and parentage testing of domestic cat specimens (14). The system simultaneously amplifies 11 polymorphic tetranucleotide STR loci and one gender identifying sequence tagged site on the Y chromosome Sex-Determining Region Y gene (SRY gene). The STR markers have been mapped in radiation hybrid and/or genetic linkage maps of the domestic cat (15,16) and were selected for forensic analysis as they are unlinked, amplify robustly, exhibit Mendelian inheritance, and exhibit a high degree of heterozygosity in cat breeds (14).Following the standard 8.1.2.2 of the quality assurance standards for DNA analysis recommended by the DNA Advisory Board (DAB) (17) and recommendations made for animal DNA forensic and identity testing (18), developmental validation studies were conducted on the cat STR multiplex typing system. These validation studies are required prior to the application of a new genetic forensic typing system to analysis of evidentiary samples, to ensure the accuracy, precision, and reproducibility of the system.     

Multiple-locus variable-number tandem repeat analysis for typing of Staphylococcus epidermidis

We applied a high-resolution PCR-based typing method, multiple-locus variable-number tandem repeat analysis (MLVA), for discrimination of 30 multidrug-resistant clinical isolates of Staphylococcus epidermidis. The results of MLVA were congruent with results obtained by pulsed-field gel electrophoresis (PFGE). MLVA generated discrete character data, and its discriminatory capacity was comparable to that of PFGE.     

Unique short tandem repeat nucleotide sequences in Entamoeba histolytica isolates from China

A few PCR-based DNA typing methods using repetitive elements contained within both protein-coding genes and noncoding DNAs have been reported for Entamoeba histolytica over the years. The serine-rich E. histolytica protein and tRNA-linked short tandem repeats (STRs) are most commonly used to investigate the relationship between parasite genotype and E. histolytica infection outcome. Many E. histolytica infections in China have been reported; however, little genome information has been provided. In the current paper, five Chinese E. histolytica samples were reported: three amoebic liver abscess cases, one combined case and one asymptomatic case. Our study is the first to report on the DNA typing information of E. histolytica in China. We included two city, one imported, and two country cases. Sequence analysis of serine-rich protein genes confirmed the presence of seven sequence types in five isolates. The STRs amplified from the samples revealed five STR variations in the A-L, four in the N-K2, and R-R loci, three in D-A, S(TGA)-D and S-Q loci. Two country patients were found to have a different outcome of infection with the same genotypes of E. histolytica, whereas in a city case, one E. histolytica strain had led to different outcome of the infection in one patient. Analyses of the results suggest that more genome information of E. histolytica strains from China through accurate methods is needed to interpret how the parasite genome plays a role in determining the outcome of E. histolytica infections.     

Accurate typing of short tandem repeats from genome-wide sequencing data and its applications

Short tandem repeats (STRs) are implicated in dozens of human genetic diseases and contribute significantly to genome variation and instability. Yet profiling STRs from short-read sequencing data is challenging because of their high sequencing error rates. Here, we developed STR-FM, short tandem repeat profiling using flank-based mapping, a computational pipeline that can detect the full spectrum of STR alleles from short-read data, can adapt to emerging read-mapping algorithms, and can be applied to heterogeneous genetic samples (e.g., tumors, viruses, and genomes of organelles). We used STR-FM to study STR error rates and patterns in publicly available human and in-house generated ultradeep plasmid sequencing data sets. We discovered that STRs sequenced with a PCR-free protocol have up to ninefold fewer errors than those sequenced with a PCR-containing protocol. We constructed an error correction model for genotyping STRs that can distinguish heterozygous alleles containing STRs with consecutive repeat numbers. Applying our model and pipeline to Illumina sequencing data with 100-bp reads, we could confidently genotype several disease-related long trinucleotide STRs. Utilizing this pipeline, for the first time we determined the genome-wide STR germline mutation rate from a deeply sequenced human pedigree. Additionally, we built a tool that recommends minimal sequencing depth for accurate STR genotyping, depending on repeat length and sequencing read length. The required read depth increases with STR length and is lower for a PCR-free protocol. This suite of tools addresses the pressing challenges surrounding STR genotyping, and thus is of wide interest to researchers investigating disease-related STRs and STR evolution.Short tandem repeats (STRs) of 1–6 base pairs per motif constitute ∼3% of the human genome (Lander 2001). Due to the high incidence of polymerase slippage at STRs (Levinson and Gutman 1987; Abdulovic et al. 2011; Baptiste and Eckert 2012), these repeats have elevated germline mutation and polymorphism rates. After a certain threshold length, STRs are termed microsatellites (Kelkar et al. 2010; Ananda et al. 2013). The high level of polymorphism makes microsatellites attractive markers for population and conservation genetics studies (Jarne and Lagoda 1996; Sunnucks 2000; Wan et al. 2004; Kim and Sappington 2013) and for identifying individuals in forensics (Hagelberg et al. 1991; Chambers et al. 2014). Many STRs are involved in gene regulation and protein function (Li et al. 2004), with ∼17% of human genes containing STRs in their open reading frames (Gemayel et al. 2010). Although long microsatellites have attracted much attention, length alterations even within relatively short repeat tracts are sometimes associated with disease (Li et al. 2004). For instance, differences in the number of repeats at the (TG)_10-13(T)_5-9 STR located within the splicing branch/acceptor site of the CFTR gene (exon 9) can affect in-frame exon skipping and, as a result, can influence the severity of cystic fibrosis (Cuppens et al. 1990; Chu et al. 1991). The purity of STRs (the degree to which the perfect STR sequence remains uninterrupted) also has a functional effect. Interrupted STRs have lower mutation rates (Ananda et al. 2014), and this can diminish disease risk. For instance, ∼6% of Ashkenazi Jews have a T to A mutation in the APC gene (encoding for a tumor suppressor) that alters an interrupted STR (A)₃T(A)₄ into a perfect (A)₈ (Laken et al. 1997). This increases the probability of somatic frameshift mutation within the STR, leading to APC protein inactivation. As a result, Ashkenazi Jews have a higher colorectal cancer risk (Gryfe et al. 1999). Since even small changes in STR length and purity can have functional effects, accurate STR profiling is crucial.Despite the importance of STRs in evolution and disease, their accurate genotyping from next generation sequencing (NGS) data has been challenging (for review, see Treangen and Salzberg 2012). Sequencing library construction frequently includes polymerase chain reaction (PCR) steps during which a polymerase might undergo slippage at STRs, leading to amplicons that differ in length due to expansion and contraction of repeat units (Ellegren 2004; Wang et al. 2011). Additionally, base calling by NGS instruments at repetitive regions is frequently imprecise. These factors result in high sequencing errors at homopolymer runs produced by the 454 (Roche) and Illumina instruments (Balzer et al. 2010; Albers et al. 2011).From a bioinformatics perspective, if STR-containing reads are mapped in their entirety, some reads cannot be mapped because of high mismatch/indel penalties associated with STR lengths different than those at the corresponding positions in the reference genome. This obscures accurate estimation of allele frequency and underestimates the real level of STR variation in the genome. To alleviate this problem, a short-read alignment approach using nonrepetitive flanks of STR-containing reads has been proposed recently (lobSTR) (Gymrek et al. 2012). This tool has fast running time and takes into account PCR stutter noise during the genotyping stage. However, the entropy scanning implemented by lobSTR to detect STRs has low sensitivity for mononucleotide STRs and short STRs (<25 bp), which constitute a large proportion of STRs in the genome. Additionally, the allele frequency at STRs for genetically heterogeneous samples, for which a simple 1:1 ratio in allele frequency present in heterozygous diploids is not expected (e.g., for tumors, viral populations, and organelles), cannot be determined. Furthermore, lobSTR uses a fixed (embedded in the program) mapping algorithm. Novel short-read mapping and STR detection algorithms (Pellegrini et al. 2010; Lim et al. 2013) are constantly being developed; an STR-profiling tool that can be customized to incorporate emerging mapping algorithms is needed.The recently released PCR-free Illumina library preparation protocol (hereafter called “PCR−”) is expected to improve STR genotyping accuracy. The direct advantage of limiting PCR steps during NGS is the increased uniformity of the sequencing depth (Kozarewa et al. 2009). Also, this protocol eliminates duplicate reads that obscure allele frequency profiling for heterogeneous genetic samples. Importantly, the degree to which the accuracy of calling STR alleles is improved using the PCR-free protocol has not been evaluated previously. Moreover, massive amounts of data have already been generated by the NGS technology with the PCR-containing library preparation protocol (hereafter called “PCR+”), and some such data cannot be regenerated due to the scarcity of samples and/or time and cost constraints. Therefore, universal methods are urgently needed that can evaluate and correct STR errors generated by NGS technology (both PCR− and PCR+) and accommodate evolving protocols and sequencing techniques.Some efforts have been made to evaluate errors generated by NGS at STRs. For instance, errors at STRs sequenced with the PCR+ protocol vary with repeat number and motif size (Luo et al. 2012). However, an explicit quantitation of various sources of STR-related sequencing errors has been lacking, which hinders an unambiguous estimation of STR mutational properties. Indeed, as both mutation and sequencing error rates increase with STR length (Kelkar et al. 2008; Luo et al. 2012; Highnam et al. 2013), one cannot confidently decipher mutation rates without accounting for sequencing error rates. Recently, a tool to guide genotyping of STRs using informed error profiles from inbred Drosophila lines (RepeatSeq) has been released (Highnam et al. 2013). This tool utilizes reads mapped by other programs, such as BWA (Li and Durbin 2009) and Bowtie (Langmead et al. 2009), and predicts the most probable genotype at a locus based on STR motif, length, and base quality. However, RepeatSeq uses the whole-read mapping approach, which introduces a bias toward the STR length in the reference genome (Gymrek et al. 2012) and thus might obscure the true STR variation spectrum. Such biases can be accounted for by an error correction model based on the STR flank-based method.To profile the full spectrum of STR lengths in the human and other genomes, and to correct for NGS-associated STR errors, we developed STR-FM (short tandem repeat profiling using a flank-based mapping approach), a flexible pipeline for detecting and genotyping STRs from short-read sequencing data. Our pipeline can detect STRs of any length, including short ones (as short as only two repeats), includes an error-correcting module, and can incorporate any NGS mapping algorithm with paired-end mapping capability, making it adaptable to new mapping methods as they become available. Applying this pipeline, we asked the following questions. First, what are the rates and patterns of sequencing errors associated with STRs of different motif sizes (mono-, di-, tri-, and tetranucleotides), motif compositions, and repeat numbers? These were contrasted between publicly available genome-wide data sets sequenced with PCR+ and PCR− protocols and validated with in-house generated, ultradeep sequencing of plasmids harboring individual STR sequences. Second, do technical errors have different patterns from true STR mutations? Third, based on the detailed knowledge of the error profiles, what is the minimum sequencing depth required for producing reliable STR genotypes for PCR+ and PCR− protocols? As a result, we provide the scientific community with STR-FM, a reproducible and versatile pipeline for genotyping STRs that incorporates an error correction model. To illustrate the utility of STR-FM, we applied it to the completely sequenced human genomes from the Platinum Genomes Project (Ajay et al. 2011) and determined human genome-wide germline mutation rates at STRs.     

短串联重复序列基因座等位基因丢失现象的研究

目的 探讨用PowerPlex(R) 16体系短串联重复(short tandem repeat,STR)基因座分型时等位基因丢失的现象及原因.方法 分析10 642宗肯定亲权的亲子鉴定案件(涉及18 314次减数分裂),对PowerPlex(R) 16体系疑似发生等位基因丢失的样本采用IdentifilerTM体系和单基因座引物体系进行验证,分离丢失的等位基因,并进行DNA序列测定和比对.结果 在D18S51、D21S1l、FGA和TPOX等4个基因座上,共确认了8宗等位基因丢失案例.通过测序均在PowerPlex(R)16体系引物结合区检见单碱基变异,包括D18S51发生4例,其中2例重复序列上游第79位碱基G→A转换,l例下游162位(G→T颠换和l例上游74位G→C颠换;D21Sll发生2例,分别为重复序列上游第17位C→A颠换和12位A→G转换;FGA和TPOX各发生1例,分别为重复序列下游142位G→A转换和下游第198位G→A转换.等位基因丢失的总发生率为0.437×10-3.结论 引物结合区的碱基变异可能导致扩增失败,产生等位基因丢失.在怀疑发生等位基因丢失时,应采用不同的引物体系进行验证以获得准确分型.在亲子鉴定结果判别和个体识别数据交换中需对此加以重视.     

中国人群中15个短串联重复序列位点的突变研究

目的对亲子鉴定中常用的PlowerPlex16(R)系统的15个短关重复序列(short tandem repeat, STR)位点的突变现象进行研究.方法在1921例确定亲权的案例中,对PlowerPlex16(R)系统的15个STR位点的突变现象进行了分析.结果在1921例确定亲权的案例中有70例(3.644%)观察到了突变,其中1例是两个位点同时突变(D21S11 and PentaD)、1例是2个子代不同位点发生突变(D7S820 and D16S539).在3764次减数分裂中,15个STR位点共观察到有72例突变,突变率为0.128%±1.104×10-3.vWA 和D21S11的突变率最高(0.292%),TH01和TPOX位点没有发现突变.父源突变是母源突变的5倍.大多数(98.611%)突变的等位基因为一步突变,一个重复单位的增加突变与减少突变之比为1.8261.只发现1例多步突变,表现为PentaD位点的等位基因的增加2个重复单位.在PlowerPlex16(R)系统中,D8S1179、Penta D、D13S317、D16S539、D7S820、D5S818、D3S1358、TH01和 TPOX 9个位点突变率低,更适用于亲权鉴定.结论 STR位点的突变是一个较为常见的现象,常使亲子鉴定中亲权认定变得更加复杂,因此筛选突变率低的稳定STR位点对于亲子鉴定非常重要.     

Francisella tularensis strain typing using multiple-locus, variable-number tandem repeat analysis

Francisella tularensis, the etiological agent of tularemia, is found throughout the Northern hemisphere. After analyzing the F. tularensis genomic sequence for potential variable-number tandem repeats (VNTRs), we developed a multilocus VNTR analysis (MLVA) typing system for this pathogen. Variation was detected at six VNTR loci in a set of 56 isolates from California, Oklahoma, Arizona, and Oregon and the F. tularensis live vaccine strain. PCR assays revealed diversity at these loci with total allele numbers ranging from 2 to 20, and Nei's diversity index values ranging from 0.36 to 0.93. Cluster analysis identified two genetically distinct groups consistent with the current biovar classification system of F. tularensis. These findings suggest that these VNTR markers are useful for identifying F. tularensis isolates at this taxonomic level. In this study, biovar B isolates were less diverse than those in biovar A, possibly reflecting the history of tularemia in North America. Seven isolates from a recent epizootic in Maricopa County, Ariz., were identical at all VNTR marker loci. Their identity, even at a hypervariable VNTR locus, indicates a common source of infection. This demonstrates the applicability of MLVA for rapid characterization and identification of outbreak isolates. Future construction of reference databases will allow faster outbreak tracking as well as providing a foundation for deciphering global genetic relationships.     

广州地区汉族群体15个短串联重复序列基因座遗传多态性分析

目的:调查广州地区汉族人群15个短串联重复序列(short tandem repeat,STR)基因座遗传多态性分布。方法:应用荧光标记多重PCR方法和毛细管电泳技术,检测156名汉族无关个体的15个STR基因座基因型。结果:15个STR基因座的基因频率分布均符合Hardy-Weinberg平衡,15个STR遗传标记均具有高度多态性,杂合度均超过0.64,15个基因座的个体识别力在0.816～0.966之间,非父排除率在0.343～0.725之间,匹配概率在0.038～0.184之间。15个基因座的累积个体识别能力为0.999 999以上,累积非父排除率为0.999 75,累积匹配概率为8.84×10-18。结论:该15个STR基因座具有高度多态性,可用于移植术后供者植入状态的监测。     

lobSTR: A short tandem repeat profiler for personal genomes

Short tandem repeats (STRs) have a wide range of applications, including medical genetics, forensics, and genetic genealogy. High-throughput sequencing (HTS) has the potential to profile hundreds of thousands of STR loci. However, mainstream bioinformatics pipelines are inadequate for the task. These pipelines treat STR mapping as gapped alignment, which results in cumbersome processing times and a biased sampling of STR alleles. Here, we present lobSTR, a novel method for profiling STRs in personal genomes. lobSTR harnesses concepts from signal processing and statistical learning to avoid gapped alignment and to address the specific noise patterns in STR calling. The speed and reliability of lobSTR exceed the performance of current mainstream algorithms for STR profiling. We validated lobSTR's accuracy by measuring its consistency in calling STRs from whole-genome sequencing of two biological replicates from the same individual, by tracing Mendelian inheritance patterns in STR alleles in whole-genome sequencing of a HapMap trio, and by comparing lobSTR results to traditional molecular techniques. Encouraged by the speed and accuracy of lobSTR, we used the algorithm to conduct a comprehensive survey of STR variations in a deeply sequenced personal genome. We traced the mutation dynamics of close to 100,000 STR loci and observed more than 50,000 STR variations in a single genome. lobSTR's implementation is an end-to-end solution. The package accepts raw sequencing reads and provides the user with the genotyping results. It is written in C/C++, includes multi-threading capabilities, and is compatible with the BAM format.     

中国朝鲜族人群6个短串联重复序列位点的遗传多态性

目的 了解中国朝鲜族人群D16S539，D7S820，D13S317，CSF1PO，TPOX，TH01 6个短串联重复序列（short tandem repeat,STR)位点的遗传多态性分布，获得相应多态位点的群体遗传学数据。方法 应用PCR扩增片段长度多态性分析方法对100名无血缘关系的朝鲜族个体进行了调查。结果 D16S539基因座，观察到6个等位基因，18种基因型；D7S820基因座，观察到7个等位基因，22种基因型；D13S317基因座，观察到7个等位基因，23种基因型；CSF1PO基因座，观察到6个等位基因，16种基因型；TPOX基因座，观察到6个等位基因，11种基因型；THO1基因座，观察到5个等位基因，12种基因型。结论 6个位点等位片段多态性分布均符合Hardy-Weinberg平衡定律。且具有较高的杂合度，所得到的等位基因频率等数据可以为中国朝鲜族人群法医个体识别、亲子鉴定及遗传学研究提供依据。     

Haplotype frequencies of 17 Y-chromosomal short tandem repeat loci from the Cukurova region of Turkey

Aim

To investigate the distribution of 17 Y-short tandem repeat (STR) loci in the population of the Cukurova region of Turkey.

Methods

In the period between 2009 and 2010, we investigated the distribution of 17 Y-STRs in a sample of 249 unrelated healthy men from the Cukurova region of Turkey. Genomic DNA was extracted with InstaGene matrix and Y-STRs were determined using the AmpFISTR Yfiler PCR amplification kit. Gene and haplotype diversity values were estimated using the Arlequin software. To compare our data to other populations, population pairwise genetic distances and associated probability values were calculated using the Y Chromosome Haplotype Reference Database Web site software.

Results

At 17 Y-STR loci we detected 148 alleles. The lowest gene diversity in this region was 0.51 for DYS391 and the highest 0.95 for DYS385a/b. Haplotype diversity was 0.9997 ± 0.0004. We compared our data with haplotype data of other Turkish populations and no significant differences were found, except with Ankara population (Φ_st = 0.025, P = 0.018). Comparisons were also made with the neighboring populations using analysis of molecular variance of the Y-STR loci genetic structure and our population was nearest to Lenkoran-Azerbaijani (Φ_st = 0.012, P = 0.068) and Iranian Ahvaz population (Φ_st = 0.007, P = 0.173), followed by Greek (Φ_st = 0.026, P = 0.000) and Russian (Φ_st = 0.048, P = 0.000) population. Other countries like Portugal, Spain, Italy, Egypt, Israel (Palestinian Authority Area), and Taiwan showed a high genetic distance from our population.

Conclusion

Our study showed that Y-STR polymorphisms were a powerful discrimination tool for routine forensic applications and could be used in genealogical investigations.Human Y chromosome short tandem repeats (Y-STR) are tandemly repeated regions of 2-7 base pair units in the non-recombining region of human Y chromosome. Since human Y-STR markers are inherited without recombination, they are transmitted from father to son unchanged. This makes the study of Y chromosome polymorphisms very useful, especially in population histories, forensic applications, and paternity analysis (1,2).The haplotype frequency of Y-STRs is important in the calculation of match probability in forensic cases. It is also important in anthropology and phylogenetic studies (1-5). Y chromosome haplotypes from global populations are stored in Y Chromosome Haplotype Reference Database (YHRD) (www.yhrd.org). The International Society of Forensic Genetics recommended the use of regional Y-STR haplotype databases to verify that no population sub-structure exists before pooling data from different regions (3). Since there is a lack of Y-STR data on Turkish population in eastern Mediterranean area, we analyzed 249 unrelated Turkish men using 17 Y-STR markers including DYS19, DYS389I, DYS389II, DYS390, DYS391, DYS392, DYS393, DYS385a/b, DYS438, DYS439, DYS437, DYS448, DYS456, DYS458, DYS635, and Y-GATA H4 from the Cukurova region in the eastern Mediterranean region of Turkey.     

华北地区汉族群体15个短串联重复序列基因座遗传多态性分析

目的调查华北地区汉族人群15个短串联重复序列(shorttandemrepeat,STR)基因座遗传多态性分布和群体遗传学数据。方法应用毛细管电泳技术和五色荧光复合扩增的方法,检测597名汉族无关个体的15个STR基因座基因型。结果15个STR基因座的基因频率分布均符合Hardy-Weinberg平衡,所检测的15个STR遗传标记均具有高度多态性,杂合度均超过0.62,15个基因座的个体识别力在0.802~0.967之间,非父排除率在0.320~0·697之间,匹配概率在0.033~0.198之间。15个基因座的累积个体识别能力为0.999999以上,累积非父排除率为0.99999571,累积匹配概率为8.93×10-18。结论联合检测15个基因座可为亲缘鉴定和个体识别提供可靠的法医学证据,这15个STR基因座适用于中国人群的法医物证学检验。