Reference Grade Characterization of Polymorphisms in Full-Length HLA Class I and II Genes With Short-Read Sequencing on the ION PGM System and Long-Reads Generated by Single Molecule, Real-Time Sequencing on the PacBio Platform

Although NGS technologies fuel advances in high-throughput HLA genotyping methods for identification and classification of HLA genes to assist with precision medicine efforts in disease and transplantation, the efficiency of these methods are impeded by the absence of adequately-characterized high-frequency HLA allele reference sequence databases for the highly polymorphic HLA gene system. Here, we report on producing a comprehensive collection of full-length HLA allele sequences for eight classical HLA loci found in the Japanese population. We augmented the second-generation short read data generated by the Ion Torrent technology with long amplicon spanning consensus reads delivered by the third-generation SMRT sequencing method to create reference grade high-quality sequences of HLA class I and II gene alleles resolved at the genomic coding and non-coding level. Forty-six DNAs were obtained from a reference set used previously to establish the HLA allele frequency data in Japanese subjects. The samples included alleles with a collective allele frequency in the Japanese population of more than 99.2%. The HLA loci were independently amplified by long-range PCR using previously designed HLA-locus specific primers and subsequently sequenced using SMRT and Ion PGM sequencers. The mapped long and short-reads were used to produce a reference library of consensus HLA allelic sequences with the help of the reference-aware software tool LAA for SMRT Sequencing. A total of 253 distinct alleles were determined for 46 healthy subjects. Of them, 137 were novel alleles: 101 SNVs and/or indels and 36 extended alleles at a partial or full-length level. Comparing the HLA sequences from the perspective of nucleotide diversity revealed that HLA-DRB1 was the most divergent among the eight HLA genes, and that the HLA-DPB1 gene sequences diverged into two distinct groups, DP2 and DP5, with evidence of independent polymorphisms generated in exon 2. We also identified two specific intronic variations in HLA-DRB1 that might be involved in rheumatoid arthritis. In conclusion, full-length HLA allele sequencing by third-generation and second-generation technologies has provided polymorphic gene reference sequences at a genomic allelic resolution including allelic variations assigned up to the field-4 level for a stronger foundation in precision medicine and HLA-related disease and transplantation studies

Ninety-year-, but not single, application of phosphorus fertilizer has a major impact on arbuscular mycorrhizal fungal communities

Background and aims Arbuscular mycorrhizal (AM) fungi play a significant role in P nutrition of crops in agriculture, but P accumulation in the soil, e.g., application of P-fertilizer, generally reduces AM fungal colonization. The impact of long-term application of chemical fertilizer on AM fungal communities was investigated with respect to the time scale. Methods Soils were collected from four plots with different fertilizer management in the long-term experimental field established in 1914. Lotus japonicus was grown in the soils in a greenhouse, while Glycine max was grown in the plots in the field. DNA was extracted from their roots, and the diversity and community compositions were analyzed based on occurrence of the AM fungal phylotypes defined by sequence similarity in the LSU rDNA. Results The 90-year-application of N and K in the absence of P increased AM fungal diversity and resulted in formation of a distinctive fungal community compared with those in the other treatments. This effect was not cancelled by single application of P. Whereas the impact of balanced application of N, P, and K was ambiguous. Conclusion These observations suggest that the presence/absence of P-fertilizer has a major impact on AM fungal communities, but the action may appear only on a long time scale. Electronic supplementary material The online version of this article (doi:10.1007/s11104-012-1398-x) contains supplementary material, which is available to authorized users