Incomplete Allele Research Articles

IndelPost: harmonizing ambiguities in simple and complex indel alignments.

Small insertions and deletions (indels) in nucleotide sequence may be represented differently between mapping algorithms and variant callers, or in the flanking sequence context. Representational ambiguity is especially profound for complex indels, complicating comparisons between multiple mappings and call sets. Complex indels may additionally suffer from incomplete allele representation, potentially leading to critical misannotation of variant effect. We present indelPost, a Python library that harmonizes these ambiguities for simple and complex indels via realignment and read-based phasing. We demonstrate that indelPost enables accurate analysis of ambiguous data and can derive the correct complex indel alleles from the simple indel predictions provided by standard small variant detectors, with improved performance over a specialized tool for complex indel analysis. indelPost is freely available at: https://github.com/stjude/indelPost. Supplementary data are available at Bioinformatics online.

P056 Extending the sequences of 569 incomplete uncommon alleles

Aim The aim of this study was to complete full gene sequences for previously reported HLA class I sequences for the IPD-IMGT/HLA database. Methods We identified 569 class I alleles in ∼ 99,000 individuals typed for a U.S. donor registry that had incomplete sequences in the IPD-IMGTHLA database. The alleles had been typed by a combined Sanger SBT-SSOP workflow focused on exons 2 + 3. Single locus alleles were amplified with primers annealing in the 5’ and 3’ UTR and the sequences of exons and introns determined with an Illumina Miseq using an in-house protocol. Results Most (423, 74%) of the 569 alleles were found in a single cell. The majority of the class I full length sequences were identical to the most similar HLA allele (e.g., C*01:70 differed by only a single exon 2 nucleotide when compared to C*01:02:01:01). Two novel HLA-A alleles with exon 2/3 variation were not detected by SBT/SSO due to inability to phase key polymorphic nucleotides [A*03:150, *26:01:01G vs. A*03:01:01:01 + A*26:149 (new); A*02:01:55 + *33:03:01G vs. A*02:01:01:01 + *33:03:33 (new)]. Overall, new alleles were found in 18 (3.2%) samples; most in the targeted incomplete allele. Most (14, 78%) showed variation in intron and UTR regions. Some of the incomplete alleles (49/569) could not be characterized because the allele combination did not allow phasing of the sequences (e.g., C*07:01:01:01 + *07:27:01 had no polymorphic nucleotides within a span of 2298 bp (introns 2–7)). Conclusions Class I gene sequences outside exons 2 + 3 are conserved. Genotypes with novel alleles can be missed by typing assays that do not provide comprehensive phasing. Increasing the knowledge of full sequences for all alleles will improve the accuracy of HLA typing. Download : Download high-res image (153KB) Download : Download full-size image J.S. Ng: 4. Scientific/Medical Advisor; Company/Organization; Georgetown University. C.K. Hurley: 4. Scientific/Medical Advisor; Company/Organization; Georgetown University.

P100 Complete single mica gene sequencing-based typing using next generation sequecers

Aim The MICA gene located in the HLA gene cluster region close to HLA-B is highly polymorphic. It is associated with solid organ transplant rejection and bone marrow transplant GVHD. To test a novel Next Generation Sequencing (NGS) based MICA high resolution typing method, we used both Sanger Based Typing (SBT) and NGS in a parallel double blind study. Methods Genomic DNA samples from 54 healthy Han people in southern China were genotyped by both SBT and NGS methods. Well established MICA SBT was performed using an amplicon covering the MICA gene from exon 2 to exon 5 (2.2kb) and MICA-Seq specific software. NGS was performed with a PCR product that includes the entire length of the MICA gene from mid 5’UTR to the end of the 3’UTR (12.6 Kb). Library preparation was performed with the standard library preparation method and reagents from Omixon’s V2 Holotype HLA kits and sequenced on an Illumina MiSeq using paired-end 2x250 bp V2 chemistry. MICA typing was assigned using Omixon Twin software and GenDx’s NGSEngine. Results 2.04 million reads with an average quality score ⩾Q30 were generated. 93.3% of reads produced were high quality and used for MICA consensus assembly with an average coverage depth of 300 across the exonic regions. 26 MICA genotypes consisting of 17 unique alleles among 54 samples were identified, and were 100% concordant between NGS and SBT. MICA NGS was able to resolve all ambiguities obtained by MICA SBT. Alleles initially typed as ambiguous MICA*008:01/04 by SBT were determined to be MICA*008:01:01, *008:01:02 and *008:04. MICA*008:04 was the most frequent at 19.4% (21/108 alleles) by NGS. In addition, 4 samples typed ambiguously as MICA*002:01/MICA*008 or MICA*023/MICA*035, and 3 samples typed as MICA*002:01/MICA*010 or MICA*022/MICA*025 by SBT were resolved by NGS. Furthermore a new *008:01 allele was found with 5 differences in intron 1 and 1 difference in intron 5. This new allele was represented three times in the population of 108 allele calls. Additionally we were able the complete the sequencing of exon 1 for *009:01 and *033 and exon 6 for *007:01 and *033 alleles. Conclusions A NGS based MICA typing method has been established and is capable of resolving ambiguities observed with SBT, identifies new alleles and allows the completion of IMGT reported incomplete allele sequences.

Effects of CYP2D6 and CYP3A5 polymorphisms on tamoxifen and its metabolites in Thai breast cancer patients.

PurposeThis study aimed to determine the effects of CYP2D6 and CYP3A5 polymorphisms on the levels of tamoxifen (TAM) and its metabolites in the plasma of breast cancer patients. The protocol was designed to test the associations between CYP2D6, CYP3A5 genotypes and phenotypes (extensive metabolizer [EM], intermediate metabolizer [IM] and poor metabolizer [PM]) and TAM, N-desmethyl tamoxifen (NDMT), endoxifen (END) and 4-hydroxytamoxifen (4OHT) concentrations.Patients and methodsOne hundred and thirty-four Thai breast cancer patients from the Thai Tamoxifen Project undergoing TAM treatment who met the inclusion/exclusion criteria were recruited. Plasma samples were assessed for the concentrations of TAM and its metabolites using high-performance liquid chromatography. The data are presented as actual values and metabolic ratios (MR). The hypotheses were tested using Kruskal–Wallis or Mann–Whitney U test, including the simple main effects analysis.ResultsThe patients had stage 0–IV breast cancer. The mean age and body mass index were 51.6±11.6 years and 24.0±4.3, respectively. Also, 53.0% of them were premenopausal, 10.4% were perimenopausal and 36.6% were postmenopausal, while 23.1% were CYP2D6-EM/CYP3A5-EM and 20.9% carried only CYP2D6 and CYP3A5 incomplete alleles. The median concentrations of TAM, NDMT, END and 4OHT were 374.7 (interquartile range [IQR] 230.2) ng/mL, 1,064.9 (IQR 599.6) ng/mL, 54.5 (IQR 52.5) ng/mL and 5.0 (IQR 3.1) ng/mL, respectively. MR (TAM-NDMT) and MR (NDMT-END) were statistically different (p=0.013 and p=0.014, respectively), while MR (4OHT-END) was not statistically different within the CYP2D6 phenotype (p=0.594). MR (TAM-4OHT) was not statistically different within the CYP2D6 phenotype (p=0.079), but it was potentially different from CYP3A5-PM (p=0.056). None of the MR was statistically different within the CYP3A5 phenotype.ConclusionCYP2D6 polymorphisms appear to affect END concentration through an NDMT subpathway and potentially affect 4OHT concentrations through a 4OHT subpathway in CYP3A5-PM group.

Imputing Genotypes in Biallelic Populations from Low-Coverage Sequence Data.

Low-coverage next-generation sequencing methodologies are routinely employed to genotype large populations. Missing data in these populations manifest both as missing markers and markers with incomplete allele recovery. False homozygous calls at heterozygous sites resulting from incomplete allele recovery confound many existing imputation algorithms. These types of systematic errors can be minimized by incorporating depth-of-sequencing read coverage into the imputation algorithm. Accordingly, we developed Low-Coverage Biallelic Impute (LB-Impute) to resolve missing data issues. LB-Impute uses a hidden Markov model that incorporates marker read coverage to determine variable emission probabilities. Robust, highly accurate imputation results were reliably obtained with LB-Impute, even at extremely low (<1×) average per-marker coverage. This finding will have implications for the design of genotype imputation algorithms in the future. LB-Impute is publicly available on GitHub at https://github.com/dellaporta-laboratory/LB-Impute.

Full-length HLA-DRB1 coding sequences generated by a hemizygous RNA-SBT approach.

Currently 1582 HLA-DRB1 alleles have been identified in the IMGT/HLA database (v3.18). Among those alleles, more than 90% have incomplete allele sequences, which complicates the analysis of the functional relevance of polymorphism beyond exon 2. The polymorphic index of each individual exon of the currently known allele sequences, shows that polymorphism is present in all exons, albeit not equally abundant. Full-length HLA-DRB1 RNA sequencing identifies polymorphism of the complete coding region. Here we describe a hemizygous full-length RNA sequence-based typing (SBT) approach based on group-specific HLA-DRB1 amplification and subsequent sequencing. RNA full-length sequences can easily be accessed because of the short amplicon length (801 bp). The RNA-SBT approach was successfully validated on a panel of DRB1 alleles having fully known coding sequences according to the IMGT/HLA database, and cover all serological equivalents. Subsequently, the approach was applied on a panel of 54 alleles with incomplete allele sequences, resulting in full-length coding sequences and the identification of one new and one corrected allele. This study shows the universal applicability of the RNA-based sequencing approach to identify full-length coding sequences and to define the polymorphic content of HLA-DRB1 alleles.

Generation of high confidence HLA genotyping and consensus sequences for class I HLA loci using the NGS-based Omixon Holotype HLA typing system and the Illumina Miseq platform

Aim Generate HLA genotyping and high confidence consensus sequences for 50 samples at HLA-A, B and C loci using the Omixon Holotype HLA typing system. Methods The Omixon Holotype HLA typing kit was used to generate sequencing libraries for 50 samples for HLA-A, B and C loci, and sequenced on an Illumina MiSeq. HLA typing and consensus sequence generation was performed using Omixon Twin, GenDx NGSengine and a custom pipeline, which relies on stringently mapping reads to the genotyped alleles and using SAMtools to generate the consensus. Consensus sequences generated by each method were aligned pairwise to determine concordance using a semi-global Needleman–Wunsch algorithm. When the sequences generated by each method were concordant, sequences were aligned to the IMGT reference sequence of the genotyped allele, to annotate novel alleles and annotate the sequence of incomplete alleles. When sequences generated by each method were discordant, sequences were aligned to the genotyped allele for manual inspection and assessment of algorithm performance. Results HLA genotyping results for all 300 alleles (50 samples at 3 loci) were 100% concordant between NGSengine and Omixon Twin, after manually resolving discrepancies (6/300). Consensus sequences were generated for every allele (300) encompassing 31 unique HLA-A, 52 unique HLA-B and 31 unique HLA-C alleles. Our analysis facilitated the annotation of full gene sequences for 11 HLA alleles for which there is no complete IMGT reference sequence (A∗02:04, B∗14:03, B∗15:08:01, B∗15:30, B∗27:02:01, B∗35:04:01, B∗35:43:01, B∗39:09:01, B∗51:08:01, B∗58:02 and C∗07:18) and facilitated the discovery of two novel HLA-A alleles, with intronic substitutions. For the remaining alleles, consensus sequences were compared against the reference sequences of the genotyped alleles and occasional differences between consensus sequences and reference sequences were manually inspected to evaluate algorithm performance. It has been determined that depth of coverage is critical in phasing and base calling, which is necessary to generate credible consensus sequences. Conclusions Generation of high confidence HLA class I consensus sequences is possible using the Omixon Holotype HLA typing system provided that adequate depth of uniform coverage is obtained from sequencing. D. Monos: Other (Identify); Company/Organization; Omixon Royalties.

Breathing abnormalities in a female mouse model of Rett syndrome.

Rett syndrome (RTT) is a female neurodevelopmental disease with breathing abnormalities. To understand whether breathing defects occur in the early lives of a group of female Mecp2(+/-) mice, a mouse model of RTT, and what percentage of mice shows RTT-like breathing abnormality, breathing activity was measured by plethysmography in conscious mice. Breathing frequency variation and central apnea in a group of Mecp2(+/-) females displayed a distribution pattern similar to Mecp2(-/Y) males, while the rest resembled the wild-type mice. Similar results were obtained using the k-mean clustering statistics analysis. With two independent methods, about 20% of female Mecp2(+/-) mice showed RTT-like breathing abnormalities that began as early as 3 weeks of age in the Mecp2(+/-) mice, and were suppressed with 3% CO2. The finding that only a small proportion of Mecp2(+/-) mice develops RTT-like breathing abnormalities suggests incomplete allele inactivation in the RTT-model Mecp2(+/-) mice.

Filling the gaps – The generation of full genomic sequences for 15 common and well-documented HLA class I alleles using next-generation sequencing technology

Many common and well-documented (CWD) HLA alleles have only been partially characterized. The DNA sequence of these incomplete alleles, as published in the IMGT/HLA database, is most often limited to exons that code for the extracellular domains of the mature protein. Here we describe the application of next-generation sequencing technology to obtain full length genomic sequence from a single long-range PCR amplicon for 15 common and well-documented HLA Class I alleles. This technology is well suited to fill in the gaps of the current HLA allele sequence database which is largely incomplete. A more comprehensive catalog of HLA allele sequences would be beneficial in the evaluation of mismatches in transplantation, studies of population genetics, the evolution of HLAs, regulatory mechanisms and HLA expression, and issues related to the genomic organization of the MHC.

Lrrk2 R1441G-related Parkinson’s disease: evidence of a common founding event in the seventh century in Northern Spain

Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene together represent the most common genetic determinant of Parkinson's disease (PD) identified to date. The vast majority of patients with LRRK2-related PD reported in the literature carry one of three pathogenic substitutions: G2019S, R1441C, or R1441G. While G2019S and R1441C are geographically widespread, R1441G is most prevalent in the Basque Country and is rare outside of Northern Spain. We sought to better understand the processes that have shaped the current distribution of R1441G. We performed a haplotype analysis of 29 unrelated PD patients heterozygous for R1441G and 85 wild-type controls using 20 markers that spanned 15.1 Mb across the LRRK2 region. Nine of the patients were of Basque origin and 20 were non-Basques. We inferred haplotypes using a Bayesian approach and utilized a maximum-likelihood method to estimate the age of the most recent common ancestor. Significant but incomplete allele sharing was observed over a distance of 6.0 Mb and a single, rare ten-marker haplotype 5.8 Mb in length was seen in all mutation carriers. We estimate that the most recent common ancestor lived 1,350 (95% CI, 1,020-1,740) years ago in approximately the seventh century. We hypothesize that R1441G originated in the Basque population and that dispersion of the mutation then occurred through short-range gene flow that was largely limited to nearby regions in Spain.

New alleles and mutational events in D12S391 and D8S1132: sequence data from an eastern German population

To investigate the DNA mutation rate and pattern in the hypervariable short tandem repeat (STR) locus D12S391 and in the locus D8S1132, samples from an eastern German population (Dresden area) were analysed. A duplex PCR was applied, using short amplification products for D12S391 (129–177 bp) and a modified reverse primer for D8S1132 (127–182 bp). The sequences of some rare and new variant alleles are described. At the locus D12S391, 13 regular and six incomplete alleles with different lengths were found, exhibiting several sequence structures. Two isolated father/child mismatches were observed in a total of 648 meioses. Novel alleles 13.1, 14.1 and 27 were discovered at the locus D8S1132. Three parent/child mismatches were found in a total of 672 meioses.