Sequence-based Alleles Research Articles

Sequenced-based French population data from 169 unrelated individuals with Verogen’s ForenSeq DNA signature prep kit

Massively Parallel Sequencing (MPS) applied to forensic genetics allows the simultaneous analysis of hundreds of genetic markers and the access to full amplicon sequences which help to increase available allele diversity. Meanwhile, sequence variation within the repeat regions represents the majority of the allele diversity, flanking regions adjacent to the repeat core provide an additional degree of variation. The forensic genetics community needs access to population data, from relevant parts of the world that contain this new sequence diversity in order to perform statistical calculations. In this study, we report sequence-based Short Tandem Repeat (STR) and identity Single Nucleotide Polymorphism (iSNPs) allele data for 169 French individuals across 58 STRs and 92 SNPs included in the Verogen ForenSeq DNA Signature Prep kit. 42 STRs out of 58 showed an increased number of alleles due to sequence variation in the repeat motif and/or the flanking regions. D9S1122 showed the largest overall gain with an increase of observed heterozygosities of almost 25 %. The combined match probability combining 27 autosomal STRs and 91 identity SNPs was 1.11E-69. Sequence-based allele frequencies included in this publication will help forensic laboratories to increase the power of discrimination for identification, kinship analysis and mixture interpretation.

Parallel sequencing of 48 Y-chromosome STR and SNP markers

Over the last decade, massively parallel sequencing (MPS) has been rapidly developed, bringing many advantages to forensic genomics. It could simultaneously target multiple genetic markers including short tandem repeats (STRs), insertion and deletion polymorphisms (InDels), and single nucleotide polymorphisms (SNPs). In this study, we developed an assay which can simultaneously sequence 22 Y-STRs and 26 Y-SNPs on the MiSeq FGx™ Forensic Genomics System (Illumina, Inc., San Diego, USA). The primers of the markers were designed using the online DesignStudio (Illumina). Subsequently, DNA libraries were established with the TruSeq Amplicon method. For the pilot study, we sequenced 25 unrelated Chinese Han males. A total of 125 sequence-based alleles were observed at the 22 Y-STR loci, with the average depth of coverage (DoC) ranging from 314.9 x to 637.7 × . For the concordant study, the sequencing results of these samples were fully consistent with genotypes called via capillary electrophoresis (CE). For the SNPs, a preferable performance was observed with the average DoC of 836.5 × . However, inter-locus imbalance was observed at some of the markers, which needs substantial improvement of the primer pool. In general, this customized Y-chromosome STR and SNP panel on the MiSeqFGx™ Forensic Genomics System can provide an effective solution for these markers on Y chromosome, which may allow more forensically relevant information to be obtained from limited forensic materials, such as those from sexual assault and rape cases. Besides, it could also facilitate the forensic practices including paternity testing, genetic determination of pedigree and inter-population migration integration.

Sequence‐based Saudi population data for the SE33 locus

A set of 87 reference samples collected from the population of Saudi Arabia were sequenced using the ForenSeq™DNA Signature Prep Kit on a MiSeq FGx™. The FASTQ files contain the sequences of the SE33 STR, but are not reported by the ForenSeq™ Universal Analysis Software (UAS). The STRait Razor software was used to recover and to report SE33 sequence‐based data for the Saudi population. Ninety-six sequence-based alleles were recovered, most of which had previously reported motif patterns. Two unreported motif patterns found in three alleles and seven novel allele sequences were reported. We also reported a single discordance between the sequence-based data and the CE data that was due to the presence of a common TTTT deletion. SE33 had 130% more sequence-based alleles; the highest number of observed sequence variants were in alleles 27.2 and 30.2, which each had 7 sequence variants. The statistical parameters emphasize the usefulness of using the sequence-based data.

Massively parallel sequence data of 31 autosomal STR loci from 496 Spanish individuals revealed concordance with CE-STR technology and enhanced discrimination power

This study reports Short Tandem Repeat (STR) sequence-based allele data from 496 Spanish individuals across 31 autosomal STR (auSTR) loci included in the Precision ID GlobalFiler™ NGS STR Panel v2: D12S391, D13S317, D8S1179, D21S11, D3S1358, D5S818, D1S1656, D2S1338, vWA, D2S441, D5S2800, D7S820, D16S539, D6S474, D12ATA63, D4S2408, D6S1043, D19S433, D14S1434, CSF1PO, D10S1248, D18S51, D1S1677, D22S1045, D2S1776, D3S4529, FGA, Penta D, Penta E, TH01 and TPOX. The sequence of each allele was aligned to the reference sequence GRCh37 (hg19) and formatted according to the guidance of the International Society for Forensic Genetics. A subset of 221 samples was evaluated for testing concordance with allele calls derived from CE-based analysis using PowerPlex Fusion 6C, and there was 99.95% allele concordance. Twenty-five out of 31 auSTR loci showed an increased number of alleles due to repeat region sequence variation and/or single nucleotide polymorphisms (SNP) residing in the flanking regions. A total of 18 loci showed increased observed heterozygosity due to sequence variation; the loci exhibiting the greatest increase were: D13S317 (12% points), D5S818 (10% points), D8S1179 (7% points), D3S1358 (7% points), and D21S11 (6% points). The combined match probability decreased from 2.022E-24 (length-based data) to 1.042E-27 (sequence-based data) for the 20 CODIS core STR loci. The combined match probability (sequence-based data) for the 31 STR loci studied was 4.777E-40. The combined typical paternity index increased from 1.118E + 12 to 8.179E + 13 using length and sequence-based data, respectively. This Spanish population study performed in the framework of the EU-funded DNASEQEX project is expected to provide STR sequence-based allele frequencies for forensic casework and support implementation of massively parallel sequencing (MPS) technology in forensic laboratories.

A nomenclature for sequence-based forensic DNA analysis

Forensic DNA analysis of casework samples using massively parallel sequencing (MPS) technology requires a system of nomenclature for uniquely labeling sequence-based alleles and artifacts. The DNA Commission of the ISFG has published considerations concerning a nomenclature format that addresses the requirement for unique labeling of sequences. Nomenclatures based on this format can be used in databasing, or communicating sequence types, but the format is lengthy for software interfaces. The sequence identifier (SID) nomenclature addresses this gap by generating short labels able to uniquely identify all sequences (allelic and artifactual) in single-source or casework profiles. Sequences in casework profiles can be uniquely labeled with only two or three SID characters, making the format compact. SID labels can be used in algorithms for identifying and filtering artifacts, and for expressing associations between artifacts and their likely parent alleles. The nomenclature is suitable for use in downstream mixture analysis by any software able to accept character values rather than numeral values. The SID nomenclature is described, and its ability to discriminate sequence-based alleles and artifacts is demonstrated, and its applicability to forensic mixture analysis is demonstrated.

Expanding beyond the current core STR loci: An exploration of 73 STR markers with increased diversity for enhanced DNA mixture deconvolution

Current approaches to mixture deconvolution of complex biological samples at times do not have the capability to resolve component contributors in DNA evidence. Additional short tandem repeat (STR) loci were sought that may improve the forensic genetic analysis of mixtures. This study presents exploratory data of a multiplex comprised of 73 highly polymorphic STRs (referred herein as the 73Plex) that were selected because of their high diversity due to sequence variation. These STRs (or a subset of them) may be considered as candidates that may augment current core markers capabilities for DNA mixture deconvolution. Population genetics analyses were performed for each locus using DNA samples from 451 individuals comprising three U.S. populations. Sequence-based heterozygosities ranged from 72% to 98%, where only two loci (D10A97 and D6A7) fell below 80%. Mixture deconvolution capabilities for two-person mixtures were assessed based on complete allele resolution per locus (i.e., four alleles observed) of pairwise mixtures using in silico methods. A subset of 20 highly informative loci (referred herein as the 20Plex) from the 73Plex was compared to the 20 CODIS core loci on all population samples with full DNA profiles for both panels (i.e., no locus dropout; n = 443). Based on proportion of loci displaying four alleles, the 20Plex outperformed the CODIS core loci with increases of 82.6% and 89.3% using length-based and sequence-based alleles, respectively. A combination of 17 STR from the 20Plex and 3 CODIS loci gave the highest capacity for resolving allelic components per locus. These data illustrate the increased value of utilizing sequenced-based alleles of additional STR loci. Furthermore, there are a number of candidate STR loci that could notably augment the current core STR loci and enhance mixture interpretation capabilities.

Paternity testing using massively parallel sequencing and the PowerSeq™ AUTO/Y system for short tandem repeat sequencing.

Massively parallel sequencing (MPS) is gaining attention as a new technology for routine forensic casework, including paternity testing. Recently released MPS multiplex panels provide many more loci compared to CE methods, plus provide sequence-based alleles that together improve the statistical power of the genetic testing. Here, an MPS system (PowerSeq™ AUTO/Y) was applied for STR sequencing in the study of first-degree STR sequence allele inheritance from families in Southern Brazil. In 29 trios (mother-child-father) analyzed, the paternity index values generally increased when data from sequence-based analysis were used in comparison to length-based data. Further, allele inconsistencies (e.g., single repeat mutation events) between child and parents could be resolved with MPS by assessing the core repeat and flanking region sequences. Lastly, the sequence information allowed for identification of isoalleles (alleles of the same size, but different sequence) to determine specific paternal and maternal inheritances. The results from this study showed advantages of implementing sequence-based analysis, MPS, in paternity testing with improved statistical calculations and a greater resolution for the trios/families tested.

Sequence-based U.S. population data for 27 autosomal STR loci

This manuscript reports Short Tandem Repeat (STR) sequence-based allele frequencies for 1036 samples across 27 autosomal STR loci: D1S1656, TPOX, D2S441, D2S1338, D3S1358, D4S2408, FGA, D5S818, CSF1PO, D6S1043, D7S820, D8S1179, D9S1122, D10S1248, TH01, vWA, D12S391, D13S317, Penta E, D16S539, D17S1301, D18S51, D19S433, D20S482, D21S11, Penta D, and D22S1045. Sequence data were analyzed by two bioinformatic pipelines and all samples have been evaluated for concordance with alleles derived from CE-based analysis at all loci. Each reported sequence includes high-quality flanking sequence and is properly formatted according to the most recent guidance of the International Society for Forensic Genetics. In addition, GenBank accession numbers are reported for each sequence, and associated records are available in the STRSeq BioProject (https://www.ncbi.nlm.nih.gov/bioproject/380127). The D3S1358 locus demonstrates the greatest average increase in heterozygosity across populations (approximately 10 percentage points). Loci demonstrating average increase in heterozygosity from 10 to 5 percentage points include (in descending order) D9S1122, D13S317, D8S1179, D21S11, D5S818, D12S391, and D2S441. The remaining 19 loci each demonstrate less than 5 percentage point increase in average heterozygosity. Discussion includes the utility of this data in understanding traditional CE results, such as informing stutter models and understanding migration challenges, and considerations for population sampling strategies in light of the marked increase in rare alleles for several of the sequence-based STR loci. This NIST 1036 data set is expected to support the implementation of STR sequencing forensic casework by providing high-confidence sequence-based allele frequencies for the same sample set which are already the basis for population statistics in many U.S. forensic laboratories.

Filipino DNA variation at 12 X-chromosome short tandem repeat markers

Demands for solving complex kinship scenarios where only distant relatives are available for testing have risen in the past years. In these instances, other genetic markers such as X-chromosome short tandem repeat (X-STR) markers are employed to supplement autosomal and Y-chromosomal STR DNA typing. However, prior to use, the degree of STR polymorphism in the population requires evaluation through generation of an allele or haplotype frequency population database. This population database is also used for statistical evaluation of DNA typing results. Here, we report X-STR data from 143 unrelated Filipino male individuals who were genotyped via conventional polymerase chain reaction-capillary electrophoresis (PCR-CE) using the 12 X-STR loci included in the Investigator® Argus X-12 kit (Qiagen) and via massively parallel sequencing (MPS) of seven X-STR loci included in the ForenSeq™ DNA Signature Prep kit of the MiSeq® FGx™ Forensic Genomics System (Illumina). Allele calls between PCR-CE and MPS systems were consistent (100% concordance) across seven overlapping X-STRs. Allele and haplotype frequencies and other parameters of forensic interest were calculated based on length (PCR-CE, 12 X-STRs) and sequence (MPS, seven X-STRs) variations observed in the population. Results of our study indicate that the 12 X-STRs in the PCR-CE system are highly informative for the Filipino population. MPS of seven X-STR loci identified 73 X-STR alleles compared with 55 X-STR alleles that were identified solely by length via PCR-CE. Of the 73 sequence-based alleles observed, six alleles have not been reported in the literature. The population data presented here may serve as a reference Philippine frequency database of X-STRs for forensic casework applications.

Development of a massively parallel sequencing assay for investigating sequence polymorphisms of 15 short tandem repeats in a Chinese Northern Han population.

Massively parallel sequencing (MPS) has been used in forensic genetics in recent years owing to several advantages, e.g. MPS can provide precise descriptions of the repeat allele structure and variation in the repeat-flanking regions, increasing the discriminating power among loci and individuals. However, it cannot be fully utilized unless sufficient population data are available for all loci. Thus, there is a pressing need to perform population studies providing a basis for the introduction of MPS into forensic practice. Here, we constructed a multiplex PCR system with fusion primers for one-directional PCR for MPS of 15 commonly used forensic autosomal STRs and amelogenin. Samples from 554 unrelated Chinese Northern Han individuals were typed using this MPS assay. In total, 313 alleles obtained by MPS for all 15 STRs were observed, and the corresponding allele frequencies ranged between 0.0009 and 0.5162. Of all 15 loci, the number of alleles identified for 12 loci increased compared to capillary electrophoresis approaches, and for the following six loci more than double the number of alleles was found: D2S1338, D5S818, D21S11, D13S317, vWA, and D3S1358. Forensic parameters were calculated based on length and sequence-based alleles. D21S11 showed the highest heterozygosity (0.8791), discrimination power (0.9865), and paternity exclusion probability in trios (0.7529). The cumulative match probability for MPS was approximately 2.3157 × 10-20 .

Sequence-based US population data for the SE33 locus.

A set of 1036 U.S. Population Samples were sequenced using the Illumina ForenSeq DNA Signature Prep Kit. This sample set has been highly characterized using a variety of marker systems for human identification. The FASTQ files obtained from a ForenSeq DNA Signature Prep Kit experiment include several STR loci that are not reported in the associated software. These include SE33, DXS8377, DXS10148, DYS456, and DYS461. The sequence variation within the autosomal STR marker SE33 was evaluated using a customized bioinformatic approach to identify and characterize the locus in the 1036 data set. The analysis identified 53 unique alleles by length and 264 by sequence. An additional 10 alleles were detected when selected extended flanking regions were examined to resolve discordances. Allele frequencies and SE33 sequence motif patterns are reported for the 1036 data set. The comparison of numerical allele calls derived from sequence data to the allele calls obtained from commercial capillary electrophoresis-based STR typing kits resulted in 100% concordance, after manual data review and confirmation sequencing of three flanking region deletions. The analysis of this data set involved significant manual sequence curation and information support from length-based genotypes to ensure high confidence in the sequence-based allele calls. The challenges of interpreting the sequence data for SE33 consisted of high sequence noise, allele-size dependent variance in coverage, and heterozygote imbalance. As allele length increased, sequence depth of coverage and quality decreased at the terminal end. Accordingly, heterozygous genotype imbalance increased in proportion to increased distance between alleles.

A phylogenetic framework facilitates Y-STR variant discovery and classification via massively parallel sequencing

Short tandem repeats on the male-specific region of the Y chromosome (Y-STRs) are permanently linked as haplotypes, and therefore Y-STR sequence diversity can be considered within the robust framework of a phylogeny of haplogroups defined by single nucleotide polymorphisms (SNPs). Here we use massively parallel sequencing (MPS) to analyse the 23 Y-STRs in Promega’s prototype PowerSeq™ Auto/Mito/Y System kit (containing the markers of the PowerPlex® Y23 [PPY23] System) in a set of 100 diverse Y chromosomes whose phylogenetic relationships are known from previous megabase-scale resequencing. Including allele duplications and alleles resulting from likely somatic mutation, we characterised 2311 alleles, demonstrating 99.83% concordance with capillary electrophoresis (CE) data on the same sample set. The set contains 267 distinct sequence-based alleles (an increase of 58% compared to the 169 detectable by CE), including 60 novel Y-STR variants phased with their flanking sequences which have not been reported previously to our knowledge. Variation includes 46 distinct alleles containing non-reference variants of SNPs/indels in both repeat and flanking regions, and 145 distinct alleles containing repeat pattern variants (RPV). For DYS385a,b, DYS481 and DYS390 we observed repeat count variation in short flanking segments previously considered invariable, and suggest new MPS-based structural designations based on these. We considered the observed variation in the context of the Y phylogeny: several specific haplogroup associations were observed for SNPs and indels, reflecting the low mutation rates of such variant types; however, RPVs showed less phylogenetic coherence and more recurrence, reflecting their relatively high mutation rates. In conclusion, our study reveals considerable additional diversity at the Y-STRs of the PPY23 set via MPS analysis, demonstrates high concordance with CE data, facilitates nomenclature standardisation, and places Y-STR sequence variants in their phylogenetic context.

Genetic analysis of Southern Brazil subjects using the PowerSeq™ AUTO/Y system for short tandem repeat sequencing

With the advent of Next-Generation Sequencing technology, sequencing of short tandem repeats (STRs) allows for a more detailed analysis when compared to size-based fragment methods (capillary electrophoresis-CE). The implementation of high-throughput sequencing can help uncover deeper genetic diversities of different populations. Subjects from the South region of Brazil present a particular and more homogeneous ancestry background when compared to other regions of the country. Both autosomal and Y- STRs have been analyzed in these individuals; however, all analyses published to date encompass data from CE-based fragment analysis. In this study, a genetic analysis of 59 individuals from Southern Brazil was performed on STR sequences. Forensically relevant STRs were PCR-enriched using a prototype of the PowerSeq™ AUTO/Y system (Promega Corp.). Next-generation sequencing was performed on an Illumina MiSeq instrument. The raw data (FASTQ files) were processed using a custom designed sequence processing tool, Altius. Isoalleles, which are sequence-based allelic variants that do not differ in length, were observed in nine autosomal and in six Y- STRs from the core global forensic marker set. The number of distinctive alleles based on sequence was higher when compared to those based on length, 37.3% higher in autosomal STRs and 13.8% higher in Y-STRs. The most polymorphic autosomal locus was D12S391, which presented 38 different sequence-based alleles. Among the loci in the Y chromosome, DYS389II presented the highest number of isoalleles. In comparison to CE analysis, Observed and Expected Heterozygosity, Polymorphic Information Content (PIC) and Genetic Diversity also presented higher values when the alleles were analyzed based on their sequence. For autosomal loci, Polymorphic Information Content (PIC) was 2.6% higher for sequence-based data. Diversity was 9.3% and 6.5% higher for autosomal and Y markers, respectively. In the analysis of the repeat structures for the STR loci, a new allele variant was found for allele 18 in the vWA locus. The STR flanking regions were also further investigated and sixteen variations were observed at nine autosomal STR loci and one Y-STR locus. The results obtained in this study demonstrate the importance of genetic analysis based on sequencing and highlight the diversity of the South Brazilian population when characterized by STR sequencing.

Performing a BLAST search of the STRSeq BioProject

At the 27th ISFG meeting in Seoul, we presented STRSeq, a catalog of high-confidence sequences at forensic short tandem repeat (STR) loci targeted in commercially available sequencing assays. This project was initiated to facilitate the description of sequence-based alleles, providing a framework for communication among laboratories. The STRSeq catalog is maintained at NCBI, in the form of GenBank records organized in a BioProject hierarchy. Herein we describe a method by which a user can query this catalog using the NCBI BLAST application.

STRSeq: A catalog of sequence diversity at human identification Short Tandem Repeat loci

The STR Sequencing Project (STRSeq) was initiated to facilitate the description of sequence-based alleles at the Short Tandem Repeat (STR) loci targeted in human identification assays. This international collaborative effort, which has been endorsed by the ISFG DNA Commission, provides a framework for communication among laboratories. The initial data used to populate the project are the aggregate alleles observed in targeted sequencing studies across four laboratories: National Institute of Standards and Technology (N=1786), Kings College London (N=1043), University of North Texas Health Sciences Center (N=839), and University of Santiago de Compostela (N=944), for a total of 4612 individuals. STRSeq data are maintained as GenBank records at the U.S. National Center for Biotechnology Information (NCBI), which participates in a daily data exchange with the DNA DataBank of Japan (DDBJ) and the European Nucleotide Archive (ENA). Each GenBank record contains the observed sequence of a STR region, annotation (“bracketing”) of the repeat region and flanking region polymorphisms, information regarding the sequencing assay and data quality, and backward compatible length-based allele designation. STRSeq GenBank records are organized within a BioProject at NCBI (https://www.ncbi.nlm.nih.gov/bioproject/380127), which is sub-divided into: commonly used autosomal STRs, alternate autosomal STRs, Y-chromosomal STRs, and X-chromosomal STRs. Each of these categories is further divided into locus-specific BioProjects. The BioProject hierarchy facilitates access to the GenBank records by browsing, BLAST searching, or ftp download. Future plans include user interface tools at strseq.nist.gov, a pathway for submission of additional allele records by laboratories performing population sample sequencing and interaction with the STRidER web portal for quality control (http://strider.online).

Genetic analysis of the Yavapai Native Americans from West-Central Arizona using the Illumina MiSeq FGx™ forensic genomics system

Forensically-relevant genetic markers were typed for sixty-two Yavapai Native Americans using the ForenSeq™ DNA Signature Prep Kit.These data are invaluable to the human identity community due to the greater genetic differentiation among Native American tribes than among other subdivisions within major populations of the United States. Autosomal, X-chromosomal, and Y-chromosomal short tandem repeat (STR) and identity-informative (iSNPs), ancestry-informative (aSNPs), and phenotype-informative (pSNPs) single nucleotide polymorphism (SNP) allele frequencies are reported. Sequence-based allelic variants were observed in 13 autosomal, 3 X, and 3 Y STRs. These observations increased observed and expected heterozygosities for autosomal STRs by 0.081±0.068 and 0.073±0.063, respectively, and decreased single-locus random match probabilities by 0.051±0.043 for 13 autosomal STRs. The autosomal random match probabilities (RMPs) were 2.37×10–26 and 2.81×10–29 for length-based and sequence-based alleles, respectively. There were 22 and 25 unique Y-STR haplotypes among 26 males, generating haplotype diversities of 0.95 and 0.96, for length-based and sequencebased alleles, respectively. Of the 26 haplotypes generated, 17 were assigned to haplogroup Q, three to haplogroup R1b, two each to haplogroups E1b1b and L, and one each to haplogroups R1a and I1. Male and female sequence-based X-STR random match probabilities were 3.28×10–7 and 1.22×10–6, respectively. The average observed and expected heterozygosities for 94 iSNPs were 0.39±0.12 and 0.39±0.13, respectively, and the combined iSNP RMP was 1.08×10–32. The combined STR and iSNP RMPs were 2.55×10–58 and 3.02×10–61 for length-based and sequence-based STR alleles, respectively. Ancestry and phenotypic SNP information, performed using the ForenSeq™ Universal Analysis Software, predicted black hair, brown eyes, and some probability of East Asian ancestry for all but one sample that clustered between European and Admixed American ancestry on a principal components analysis. These data serve as the first population assessment using the ForenSeq™ panel and highlight the value of employing sequence-based alleles for forensic DNA typing to increase heterozygosity, which is beneficial for identity testing in populations with reduced genetic diversity.

Identification and validation of novel alleles of rice blast resistant gene Pi54, and analysis of their nucleotide diversity in landraces and wild Oryza species

Rice blast is a devastating fungal disease, which limits rice production severely. To identify novel alleles of blast resistant gene, Pi54, and to understand its diversity exist among wild Oryza species and landraces sequence-based allele mining strategy was employed. In this study, sixteen Pi54 alleles were sequenced from landraces and wild Oryza species which have genome ranging from AA to EE. Overall analysis confirmed that the alleles derived from wild species had more divergence than the landraces. Among all wild Oryza alleles, Pi54 aus derived from O. australiensis, showed the highest diversity. Comparison of protein domains revealed that LRR region had more variations than NBS region. Haplo-groups among the ecotypes were analyzed based on their Pi54 sequence. Interestingly, four different haplo-groups were identified, that were supported by phylogeny analysis. Among 16 analyzed alleles, two alleles, Pi54 ab, Pi54 btj derived from landraces-Amana Bavo and Boha Thulasi Joha showed superior phenotypic reaction than the reference allele (Pi54 Tetep), and these alleles were validated by allelism test. These alleles have potential application in blast resistance breeding programs and identified SNPs and Indel among the alleles can be useful in development of allele specific markers. The present study helped to gain insights into the evolutionary adaptation of the resistant gene and its allelic distribution and diversity among the Oryza species.

Correction: Corrigendum: Identification of novel alleles of the rice blast resistance gene Pi54

Rice blast is one of the most devastating rice diseases and continuous resistance breeding is required to control the disease. The rice blast resistance gene Pi54 initially identified in an Indian cultivar confers broad-spectrum resistance in India. We explored the allelic diversity of the Pi54 gene among 885 Indian rice genotypes that were found resistant in our screening against field mixture of naturally existing M. oryzae strains as well as against five unique strains. These genotypes are also annotated as rice blast resistant in the International Rice Genebank database. Sequence-based allele mining was used to amplify and clone the Pi54 allelic variants. Nine new alleles of Pi54 were identified based on the nucleotide sequence comparison to the Pi54 reference sequence as well as to already known Pi54 alleles. DNA sequence analysis of the newly identified Pi54 alleles revealed several single polymorphic sites, three double deletions and an eight base pair deletion. A SNP-rich region was found between a tyrosine kinase phosphorylation site and the nucleotide binding site (NBS) domain. Together, the newly identified Pi54 alleles expand the allelic series and are candidates for rice blast resistance breeding programs.

Identification of novel alleles of the rice blast resistance gene Pi54.

Rice blast is one of the most devastating rice diseases and continuous resistance breeding is required to control the disease. The rice blast resistance gene Pi54 initially identified in an Indian cultivar confers broad-spectrum resistance in India. We explored the allelic diversity of the Pi54 gene among 885 Indian rice genotypes that were found resistant in our screening against field mixture of naturally existing M. oryzae strains as well as against five unique strains. These genotypes are also annotated as rice blast resistant in the International Rice Genebank database. Sequence-based allele mining was used to amplify and clone the Pi54 allelic variants. Nine new alleles of Pi54 were identified based on the nucleotide sequence comparison to the Pi54 reference sequence as well as to already known Pi54 alleles. DNA sequence analysis of the newly identified Pi54 alleles revealed several single polymorphic sites, three double deletions and an eight base pair deletion. A SNP-rich region was found between a tyrosine kinase phosphorylation site and the nucleotide binding site (NBS) domain. Together, the newly identified Pi54 alleles expand the allelic series and are candidates for rice blast resistance breeding programs.

Comparable Survival and Transplant-Related Mortality Following Allogeneic Hematopoietic Cell Transplantation from HLA Allele-Matched Unrelated and HLA-Identical Sibling Donors.

Comprehensive analyses of unrelated donor (URD) and recipient HLA-matching for allogeneic hematopoietic cell transplantation (AHCT) have demonstrated better outcomes when allele typing is performed using high-resolution nucleotide sequence-based techniques. To evaluate survival following myeloablative AHCT using allele-level HLA-matched URD as compared with HLA-identical sibling donors, we analyzed outcomes for 430 patients treated at our center between March 1991 and April 2005. Sequence-based allele typing was retrospectively performed for HLA-A, B, C, DR and DQ when not done at time of AHCT for URD (n = 124; 29%) and non-sibling related donors (n = 19; 4%). Donors were HLA-identical siblings (n = 276; 64%), HLA allele-matched URD (n = 52; 12%), single HLA-locus mismatched donors (n = 52; 12%), or > 1 locus mismatched donors (n = 50; 12%). The median age at transplant was 23.4 years (range: 0.2 – 61). The most common diagnoses were AML (n = 107; 25%), CML (n = 90; 21%), ALL (n = 86; 20%) and MDS (n = 50; 12%). Total body irradiation-based preparative regimens were used for 283 patients (66%). Bone marrow (BM) was the graft for 388 patients (90%) and GCSF-mobilized peripheral blood stem cells (PBSC) for the remaining 42 (10%). Graft-versus-host disease (GVHD) prophylaxes were cyclosporine and methotrexate (n = 327; 76%), long methotrexate (n = 42; 10%), T-cell depletion (n = 19; 4%), or other regimens (n = 42; 10%). With a median follow-up of 4.8 years (range: 0.2 – 12.1), the 5-year estimate of overall survival (OS) for the entire group was 48.2% (95% CI: 45.7 – 50.7) and transplant-related mortality (TRM) was 31.4% (95% CI: 28.8 – 34). As shown in the Table, OS and TRM were indistinguishable between AHCT performed with HLA-identical siblings compared with HLA allele-matched URD. There was also no difference in grade III – IV acute GVHD (P = .46) between these two groups whereas there was a trend towards more extensive chronic GVHD (HR 1.8; 95% CI: 0.9 – 3.6; P = 0.12) for the URD recipients. Using a multivariate analysis to adjust for advanced disease, age (> vs ≤ 30 years), graft (BM vs PBSC) and female-to-male gender mismatch, there remained no difference in OS between HLA-identical siblings and HLA allele-matched URD (P = 0.67). These results demonstrate that key outcomes (OS, TRM, and severe acute GVHD) are equivalent in recipients of grafts from either allele-level 10/10 HLA-matched URD or HLA-identical siblings.Overall SurvivalTRMNumberHazard Ratio95% CIP valueHazard Ratio95% CIP valueHLA-ID Sibling2761--1--HLA-ID URD521.10.7 – 1.70.670.80.4 – 1.60.581 Locus MM521.30.9 – 2.00.191.40.8 – 2.40.25> 1 Locus MM502.01.4 – 2.9< 0.0012.61.7 – 4.1< 0.001