Large-scale MHC class II genotyping of a wild lemur population by next generation sequencing

The critical role of major histocompatibility complex (MHC) genes in disease resistance, along with their putative function in sexual selection, reproduction and chemical ecology, make them an important genetic system in evolutionary ecology. Studying selective pressures acting on MHC genes in the wild nevertheless requires population-wide genotyping, which has long been challenging because of their extensive polymorphism. Here, we report on large-scale genotyping of the MHC class II loci of the grey mouse lemur (Microcebus murinus) from a wild population in western Madagascar. The second exons from MHC-DRB and -DQB of 772 and 672 individuals were sequenced, respectively, using a 454 sequencing platform, generating more than 800,000 reads. Sequence analysis, through a stepwise variant validation procedure, allowed reliable typing of more than 600 individuals. The quality of our genotyping was evaluated through three independent methods, namely genotyping the same individuals by both cloning and 454 sequencing, running duplicates, and comparing parent–offspring dyads; each displaying very high accuracy. A total of 61 (including 20 new) and 60 (including 53 new) alleles were detected at DRB and DQB genes, respectively. Both loci were non-duplicated, in tight linkage disequilibrium and in Hardy–Weinberg equilibrium, despite the fact that sequence analysis revealed clear evidence of historical selection. Our results highlight the potential of 454 sequencing technology in attempts to investigate patterns of selection shaping MHC variation in contemporary populations. The power of this approach will nevertheless be conditional upon strict quality control of the genotyping data.     

Efficacy and limits of genotyping low copy number (LCN) DNA samples by multiplex PCR of STR loci

In this study, we have evaluated the efficacy and the validity of the AmpFISTR SGM plus multiplex PCR typing system when Low Copy Number (LCN) amounts of DNA are processed. The characteristics of SGM plus profiles produced under LCN conditions were studied on the basis of heterozygote balance, between loci balance and stutter proportion based on profiles that were obtained from a variety of mock casework samples. These experiments clearly showed that LCN DNA profiles carry their own characteristic features, which must be taken into account during interpretation. Herewith, we confirmed the data of recent other studies that a comprehensive interpretation strategy is dependent upon multiple replication of the PCR using the same extract together with the proper use of extraction and amplification controls. The limitations of LCN DNA analysis were further studied in a series of single cell PCR experiments using an amplification regime of 34 PCR cycles. The allele dropout phenomenon was demonstrated to its full extent when single cells were analysed. However, the "consensus profile" which was obtained from separate single cell PCR experiments matched the actual profile of the cell donor. Single cell PCR experiments also showed that a further increase of the number of PCR cycles did not result in enhanced sensitivity and had a highly negative effect on the balance of this multiplex PCR system which hampered correct interpretation of the profile. Also, the potential of LCN typing in analysing mixtures of DNA was investigated. It was clearly shown that LCN typing had no advantages over 28 cycles amplification in the detection of the minor component of DNA-mixtures. In addition to the 34 cycles PCR amplification regime, the utility of a new approach that involved reamplification of the 28 cycle SGM plus PCR products with an extra 6 PCR cycles after the addition of fresh AmpliTaq Gold DNA Polymerase was investigated. This approach provides the scientist with an extra typing result that enhances the reliability of the consensus profile, which is commonly retrieved from two separate 34 cycle PCR results. Furthermore, the 28 + 6 cycles approach may be used to screen LCN samples for their potential to produce a 34 PCR cycle profile. Finally and as a last resort the 28 + 6 cycles approach can be used in those cases where no further extract from the crime sample is available. Finally, the potential of LCN typing was demonstrated in typing samples from non-probative and actual casework examples. From a high proportion of samples that failed to demonstrate SGM plus typing results using the standard protocol of 28 cycles, at least partial profiles could be obtained after LCN methods were used. For example, LCN typing was applied in a case where 10-year old samples from bones and teeth that were retrieved from a mass grave had to be identified. This study resulted in the positive identification of a number of victims by comparing the LCN DNA profiles with the profiles from putative relatives. The value of LCN DNA typing was further demonstrated in a strangulation case. The throat of the victim was sampled and only after 34 PCR cycles were we able to reveal that the evidential sample contained a distinct mixture of the victim's own DNA and the DNA of the defendant.     

Design of a multiplex PCR for genotyping 16 short tandem repeats in degraded DNA samples

The molecular genotyping of individuals and reconstruction of kinship through short and high polymorphic DNA markers, so-called short tandem repeats (STR), has become an important and efficient method in anthropology and forensic science. The here introduced experimental design describes a multiplex PCR capable of simultaneously amplifying 16 STRs and the sex determinant locus amelogenin in a short fragment lengths range from 84 bp to 275 bp. Thus, the design depends predominantly on the routines for DNA typing of historical samples with highly degraded ancient DNA. It is shown, that the newly designed multiplex PCR is suitable for successful typing of both forensic and historical material.     

7个Y-STR基因座荧光标记复合分型及在中国汉族群体中遗传多态性的研究

建立了FAM（蓝色荧光）标记的DYS456、DYS464a/b/c/d和DYS527a/b和JOE(绿色荧光)标记的DYS531、DYS709、DYS448和DYS522 7个Y染色体STR基因座(相当于11个位点)复合扩增分型体系。利用ABI3100遗传分析仪调查中国广东汉族151例和河南106例汉族无关男性个体的遗传多态性分布, 并探讨在法医学中的应用价值。结果显示, 此方法能作出正确分型的最低基因组DNA量为0.02 ng; 在女性成份为男性成份150倍的混合DNA样本(总DNA量为160 ng)中能正确检出全部7个Y-STR基因座的基因型。 广东和河南2个汉族男性群体中, 观察到的单体型分别有150、105种, 其中仅观察到1次的单体型分别有149、104种, 单体型多样性(HD)分别为0.999912、0.999820; 这组Y-STR单体型在两个群体中的分布差异有统计学显著意义(秩和检验: P<0.001)。此7个Y-STR基因座多态性分析适用于法医学实践和人类进化的研究。     

Detection of four important Eimeria species by multiplex PCR in a single assay

The oocysts of some of the recognized species of chicken coccidiosis are difficult to distinguish morphologically. Diagnostic laboratories are increasingly utilizing DNA-based technologies for the specific identification of Eimeria species. This study reports a multiplex polymerase chain reaction (PCR) assay based on internal transcribed spacer-1 (ITS-1) for the simultaneous diagnosis of the Eimeria tenella, Eimeria acervulina, Eimeria maxima, and Eimeria necatrix species, which infect domestic fowl. Primer pairs specific to each species were designed in order to generate a ladder of amplification products ranging from 20 to 25 bp, and a common optimum annealing temperature for these species was determined to be 52.5 °C. Sensitivity tests were performed for each species, showing a detection threshold of 1–5 pg. All the species were amplified homogeneously, and a homogenous band ladder was observed, indicating that the assay permitted the simultaneous detection of all the species in a single-tube reaction. In the phylogenic study, there was a clear species clustering, which was irrespective of geographical location, for all the ITS-1 sequences used. This multiplex PCR assay represents a rapid and potential cost-effective diagnostic method for the detection of some key Eimeria species that infect domestic fowl.     

Utilizing next generation sequencing to characterize microsatellite loci in a tropical aquatic plant species Cryptocoryne cordata var. cordata (Araceae)

Cryptocoryne cordata var. cordata (2n = 34) is an aquatic plant species distributed from the southern part of Peninsular Thailand through the Malay Peninsula. It propagates both sexually and asexually via stolons. The current study is aimed at developing nuclear microsatellite markers for the species using next generation sequencing (Roche 454 pyrosequencing) from genomic DNA. A total of 41,653 reads was generated, of which 3636 fragments contained at least one repeat motif. Seventy two primer sets in the flanking region of dinucleotide, trinucleotide and tetranucleotides repeat motifs were designed and tested for efficiency in polymerase chain reaction amplification. Using these primer sets, 11 new microsatellite marker loci were successfully amplified with unambiguous polymorphic alleles exhibited across 30 individuals examined. The number of alleles per locus ranged from 2 to 6, while observed and expected heterozygosity ranged from 0.8190 to 1.0000 and 0.5401 to 0.7548, respectively. Genotype frequencies at all loci departed significantly from Hardy–Weinberg equilibrium. Linkage disequilibrium was not detected between any pair of loci. Cross-species amplification was successful across a panel of ten Cryptocoryne species. The markers described in this study will be useful for evaluating genetic diversity within and between populations, levels of gene flow, and the population dynamics of clones. They will be of further value in the development of effective conservation programs for Cryptocoryne species.     

Microsatellite-directed selection of breeders for the next backcross generation by using a minimal number of loci

To establish a minimal number of markers for direct selection of candidate mice used for the next mating to produce congenic mice, recombination frequencies of 53 microsatellite loci on chromosomes (chr.) 1 and 19 were examined using 41 N2 mice: the donor strain was BALB/c, and recipient strain was C57BL/6J (B6J) or C57BL/6N (B6N). These markers were spaced at 0.1 to 24.2 centimorgans (cM). Among the 41 mice, B6/B6 homozygosity ranged from 18 to 24 animals (mean, 20; 2 standard deviations, 1.36) for a given locus. There was no difference in recombination frequency between chr. 1 and 19. The recombination frequency of B6J was higher than that of B6N (P < 0.05). Various densities of markers, 10 (5 markers/chr.), 8 (4 markers/chr.), and 6 (3 markers/chr.) spaced at 12.0 to 29.3, 9.0 to 45.0, and 24.5 to 53.0 cM, respectively, were selected from the 53 markers, and homozygosity was compared in each mouse. In mice with decreased homozygosity when tested using 53 markers, homozygosity differed depending on the density of the markers. The results suggested that 3 markers/chr. are sufficient for selection of the highest percentages of homozygosity but are not suitable to define mice with lower percentages of homozygosity.     

Pool‐hmm: a Python program for estimating the allele frequency spectrum and detecting selective sweeps from next generation sequencing of pooled samples

Due to its cost effectiveness, next generation sequencing of pools of individuals (Pool‐Seq) is becoming a popular strategy for genome‐wide estimation of allele frequencies in population samples. As the allele frequency spectrum provides information about past episodes of selection, Pool‐seq is also a promising design for genomic scans for selection. However, no software tool has yet been developed for selection scans based on Pool‐Seq data. We introduce Pool‐hmm, a Python program for the estimation of allele frequencies and the detection of selective sweeps in a Pool‐Seq sample. Pool‐hmm includes several options that allow a flexible analysis of Pool‐Seq data, and can be run in parallel on several processors. Source code and documentation for Pool‐hmm is freely available at https://qgsp.jouy.inra.fr/ .     

Exome sequencing followed by large-scale genotyping fails to identify single rare variants of large effect in idiopathic generalized epilepsy

Idiopathic generalized epilepsy (IGE) is a complex disease with high heritability, but little is known about its genetic architecture. Rare copy-number variants have been found to explain nearly 3% of individuals with IGE; however, it remains unclear whether variants with moderate effect size and frequencies below what are reliably detected with genome-wide association studies contribute significantly to disease risk. In this study, we compare the exome sequences of 118 individuals with IGE and 242 controls of European ancestry by using next-generation sequencing. The exome-sequenced epilepsy cases include study subjects with two forms of IGE, including juvenile myoclonic epilepsy (n = 93) and absence epilepsy (n = 25). However, our discovery strategy did not assume common genetic control between the subtypes of IGE considered. In the sequence data, as expected, no variants were significantly associated with the IGE phenotype or more specific IGE diagnoses. We then selected 3,897 candidate epilepsy-susceptibility variants from the sequence data and genotyped them in a larger set of 878 individuals with IGE and 1,830 controls. Again, no variant achieved statistical significance. However, 1,935 variants were observed exclusively in cases either as heterozygous or homozygous genotypes. It is likely that this set of variants includes real risk factors. The lack of significant association evidence of single variants with disease in this two-stage approach emphasizes the high genetic heterogeneity of epilepsy disorders, suggests that the impact of any individual single-nucleotide variant in this disease is small, and indicates that gene-based approaches might be more successful for future sequencing studies of epilepsy predisposition.     

Application of a multiplex PCR genotyping assay of 31 red blood cell antigens for establishment of a panel of reference DNA in China

DNA based blood group genotyping has been widely used in clinical blood transfusions, and a number of different molecular blood group testing methods have been developed, including the detection of single nucleotide polymorphism (SNP) and genomic DNA sequencing. As the molecular bases of blood groups can differ widely between ethnic groups, a set of reference DNAs, especially for the Chinese population, is required for the development and validation of these methods, and for their optimal use in routine practice in China. In this study, a total of 100 DNA samples obtained from 60 established cell lines and 40 Chinese blood donors were typed for 31 red blood cell antigens of 13 blood group systems using a multiplex polymerase chain reaction (PCR) assay. Finally, nine DNA samples were selected to establish a panel of reference DNA that included M, N, S, s, Mur, Lu^a, Lu^b, Au^a, Au^b, K, k, Fy^a, Fy^b, Jk^a, Jk^b, Di^a, Di^b, Sc1, Sc2, Do^a, Do^b, Co^a, Co^b, Kn^a, Kn^b, In^b, Vel antigens and Fy (a-b-) null phenotype.     

Genome-Tagged Amplification (GTA): a PCR-based method to prepare sample-tagged amplicons from hundreds of individuals for next generation sequencing

Sampling the sequence of a relatively small fraction of the genome in large numbers of individuals is an important objective for population genetics and association genetics approaches. However, currently available ‘sequence capture’ methods either require expensive instrumentation or have problems dealing with high sample numbers and relatively small target sizes. We have developed Genome-Tagged Amplification (GTA) as a flexible PCR-based method for preparing pools of hundreds of amplicons from hundreds of samples for next generation sequencing. The method involves tagging of genomic DNA with barcode adapters at restriction sites, followed by PCR amplification from flanking DNA. It is freely scalable for both sample number and amplicon number and has no specialized equipment requirement. An optimized protocol is presented which provides a matrix of 96 × 192 combinations of samples x amplicons, corresponding to a complete 454 Titanium run. Initially, we used 454 sequencing; however, GTA could easily be adapted to Illumina sequencing platforms as read lengths have significantly increased in this system.     

Application of a multiplex PCR for the detection of protozoan pathogens of the eastern oyster Crassostrea virginica in field samples

Populations of eastern oysters Crassostrea virginica along the east coast of North America have repeatedly experienced epizootic mass mortality due to infections by protozoan parasites, and molecular diagnostic methodologies are fast becoming more widely available for the diagnosis of protozoan diseases of oysters. In this study we applied a modified version of an existing multiplex polymerase chain reaction (PCR) for detection of the eastern oyster parasites Haplosporidium nelsoni, H. costale and Perkinsus marinus from field-collected samples. We incorporated primers for DNA quality control based on the large subunit ribosomal RNA (LSU rRNA) gene of C. virginica. The multiplex PCR (MPCR) simultaneously amplified genomic DNA of C. virginica, and cloned DNA of H. nelsoni, P. marinus and H. costale. In field trial applications, we compared the performance of the MPCR to that of the conventional diagnostic techniques of histopathological tissue examination and the Ray/Mackin fluid thioglycollate medium (RMFT) assay. A total of 530 oysters were sampled from 18 sites at 12 locations along the east coast of the United States from the Gulf of Mexico to southern New England. The modified MPCR detected 21% oysters with H. nelsoni, 2% oysters with H. costale, and 40% oysters with P. marinus infections. In comparison, histopathological examination detected H. nelsoni and H. costale infections in 6 and 0.8% oysters, respectively, and the RMFT assay detected P. marinus infection in 31% oysters. The MPCR is a more sensitive diagnostic assay for detection of H. nelsoni, H. costale, and P. marinus, and incorporation of an oyster quality control product limits false negative results.     

Costs of growing up as a subordinate sibling are passed to the next generation in blue‐footed boobies

As stresses in early development may generate costs in adult life, sibling competition and conflict in infancy are expected to diminish the reproductive value of surviving low‐status members of broods and litters. We analysed delayed costs to blue‐footed booby fledglings, Sula nebouxii, of junior status in the brood, which involves aggressive subordination, food deprivation and elevated corticosterone, but little or no deficit in size at fledging. In ten cohorts observed for up to 16 years, juniors showed no deficit in breeding success at any age, independent of lifespan, including in a sample of sibling pairs. Among females, juniors actually outreproduced seniors across the 16‐year span. However, offspring produced by juniors in the first 3 years of life were less likely to recruit into the breeding population than offspring of seniors. Since junior fledglings survive, recruit and compete as well as seniors (shown earlier), and breed as successfully as seniors across the lifespan, it appears the delayed cost of subordination is passed to offspring, and only to those few offspring produced in the first 3 years of life. These correlational results indicate that systematic competition‐related differences in developmental conditions of infant siblings can alter their reproductive value by affecting the viability of their eventual offspring.     

Development of a real-time multiplex PCR assay for the detection of multiple <Emphasis Type="Italic">Salmonella</Emphasis> serotypes in chicken samples

Background  

A real-time multiplex PCR assay was developed for the detection of multiple Salmonella serotypes in chicken samples. Poultry-associated serotypes detected in the assay include Enteritidis, Gallinarum, Typhimurium, Kentucky and Dublin. The traditional cultural method according to EN ISO 6579:2002 for the detection of Salmonella in food was performed in parallel. The real-time PCR based method comprised a pre-enrichment step in Buffered Peptone Water (BPW) overnight, followed by a shortened selective enrichment in Rappaport Vasilliadis Soya Broth (RVS) for 6 hours and subsequent DNA extraction.     

FLAG assay as a novel method for real-time signal generation during PCR: application to detection and genotyping of KRAS codon 12 mutations

Real-time signal generation methods for detection and characterization of low-abundance mutations in genomic DNA are powerful tools for cancer diagnosis and prognosis. Mutations in codon 12 of the oncogene KRAS, for example, are frequently found in several types of human cancers. We have developed a novel real-time PCR technology, FLAG (FLuorescent Amplicon Generation) and adapted it for simultaneously (i) amplifying mutated codon 12 KRAS sequences, (ii) monitoring in real-time the amplification and (iii) genotyping the exact nucleotide alteration. FLAG utilizes the exceptionally thermostable endonuclease PspGI for real-time signal generation by cleavage of quenched fluorophores from the 5′-end of the PCR products and, concurrently, for selecting KRAS mutations over wild type. By including peptide-nucleic-acid probes in the reaction, simultaneous genotyping is achieved that circumvents the requirement for sequencing. FLAG enables high-throughput, closed-tube KRAS mutation detection down to ~0.1% mutant-to-wild type. The assay was validated on model systems and compared with allele-specific PCR sequencing for screening 27 cancer specimens. Diverse applications of FLAG for real-time PCR or genotyping applications in cancer, virology or infectious diseases are envisioned.     

SimRAD: an R package for simulation‐based prediction of the number of loci expected in RADseq and similar genotyping by sequencing approaches

Application of high‐throughput sequencing platforms in the field of ecology and evolutionary biology is developing quickly with the introduction of efficient methods to reduce genome complexity. Numerous approaches for genome complexity reduction have been developed using different combinations of restriction enzymes, library construction strategies and fragment size selection. As a result, the choice of which techniques to use may become cumbersome, because it is difficult to anticipate the number of loci resulting from each method. We developed SimRAD, an R package that performs in silico restriction enzyme digests and fragment size selection as implemented in most restriction site associated DNA polymorphism and genotyping by sequencing methods. In silico digestion is performed on a reference genome or on a randomly generated DNA sequence when no reference genome sequence is available. SimRAD accurately predicts the number of loci under alternative protocols when a reference genome sequence is available for the targeted species (or a close relative) but may be unreliable when no reference genome is available. SimRAD is also useful for fine‐tuning a given protocol to adjust the number of targeted loci. Here, we outline the functionality of SimRAD and provide an illustrative example of the use of the package (available on the CRAN at http://cran.r-project.org/web/packages/SimRAD ).