Very high resolution single pass HLA genotyping using amplicon sequencing on the 454 next generation DNA sequencers: Comparison with Sanger sequencing.

Compared to Sanger sequencing, next-generation sequencing offers advantages for high resolution HLA genotyping including increased throughput, lower cost, and reduced genotype ambiguity. Here we describe an enhancement of the Roche 454 GS GType HLA genotyping assay to provide very high resolution (VHR) typing, by the addition of 8 primer pairs to the original 14, to genotype 11 HLA loci. These additional amplicons help resolve common and well-documented alleles and exclude commonly found null alleles in genotype ambiguity strings. Simplification of workflow to reduce the initial preparation effort using early pooling of amplicons or the Fluidigm Access Array™ is also described. Performance of the VHR assay was evaluated on 28 well characterized cell lines using Conexio Assign MPS software which uses genomic, rather than cDNA, reference sequence. Concordance was 98.4%; 1.6% had no genotype assignment. Of concordant calls, 53% were unambiguous. To further assess the assay, 59 clinical samples were genotyped and results compared to unambiguous allele assignments obtained by prior sequence-based typing supplemented with SSO and/or SSP. Concordance was 98.7% with 58.2% as unambiguous calls; 1.3% could not be assigned. Our results show that the amplicon-based VHR assay is robust and can replace current Sanger methodology. Together with software enhancements, it has the potential to provide even higher resolution HLA typing.

High throughput HLA genotyping using 454 sequencing and the Fluidigm Access Array™ System for simplified amplicon library preparation.

The human leukocyte antigen (HLA) class I and class II loci are the most polymorphic genes in the human genome; distinguishing the thousands of HLA alleles is challenging. Next generation sequencing of exonic amplicons with the 454 genome sequence (GS) FLX System and Conexio Assign ATF 454 software provides high resolution, high throughput HLA genotyping for eight class I and class II loci. HLA typing of potential donors for unrelated bone marrow donor registries typically uses a subset of these loci at high sample throughput and low cost per sample. The Fluidigm Access Array System enables the incorporation of 48 different multiplex identifiers (MIDs) corresponding to 48 genomic DNA samples with up to 48 different primer pairs in a microfluidic device generating 2304 parallel polymerase chain reactions (PCRs). Minimal volumes of reagents are used. During genomic PCR, in this 4-primer system, the outer set of primers containing the MID and the 454 adaptor sequences are incorporated into an amplicon generated by the inner HLA target-specific primers each containing a common sequence tag at the 5' end of the forward and reverse primers. Pools of the resulting amplicons are used for emulsion PCR and clonal sequencing on the 454 Life Sciences GS FLX System, followed by genotyping with Conexio software. We have genotyped 192 samples with 100% concordance to known genotypes using 8 primer pairs (covering exons 2 and 3 of HLA-A, B and C, and exon 2 of DRB1, 3/4/5 and DQB1) and 96 MIDs in a single GS FLX run. An average of 166 reads per amplicon was obtained. We have also genotyped 96 samples at high resolution (14 primer pairs covering exons 2, 3, and 4 of the class I loci and exons 2 of DRB1, 3/4/5, DQA1, DQB1, DPB1, and exon 3 of DQB1), recovering an average of 173 sequence reads per amplicon.

A multi-site study using high-resolution HLA genotyping by next generation sequencing.

The high degree of polymorphism at human leukocyte antigen (HLA) class I and class II loci makes high-resolution HLA typing challenging. Current typing methods, including Sanger sequencing, yield ambiguous typing results because of incomplete genomic coverage and inability to set phase for HLA allele determination. The 454 Life Sciences Genome Sequencer (GS FLX) next generation sequencing system coupled with conexio atf software can provide very high-resolution HLA genotyping. High-throughput genotyping can be achieved by use of primers with multiplex identifier (MID) tags to allow pooling of the amplicons generated from different individuals prior to sequencing. We have conducted a double-blind study in which eight laboratory sites performed amplicon sequencing using GS FLX standard chemistry and genotyped the same 20 samples for HLA-A, -B, -C, DPB1, DQA1, DQB1, DRB1, DRB3, DRB4, and DRB5 (DRB3/4/5) in a single sequencing run. The average sequence read length was 250 base pairs and the average number of sequence reads per amplicon was 672, providing confidence in the allele assignments. Of the 1280 genotypes considered, assignment was possible in 95% of the cases. Failure to assign genotypes was the result of researcher procedural error or the presence of a novel allele rather than a failure of sequencing technology. Concordance with known genotypes, in cases where assignment was possible, ranged from 95.3% to 99.4% for the eight sites, with overall concordance of 97.2%. We conclude that clonal pyrosequencing using the GS FLX platform and CONEXIO ATF software allows reliable identification of HLA genotypes at high resolution.

High-resolution, high-throughput HLA genotyping by next-generation sequencing.

The human leukocyte antigen (HLA) class I and class II loci are the most polymorphic genes in the human genome. Hematopoietic stem cell transplantation requires allele-level HLA typing at multiple loci to select the best matched unrelated donors for recipient patients. In current methods for HLA typing, both alleles of a heterozygote are amplified and typed or sequenced simultaneously, often making it difficult to unambiguously determine the sequence of the two alleles. Next-generation sequencing methods clonally propagate in parallel millions of single DNA molecules, which are then also sequenced in parallel. Recently, the read lengths obtainable by one such next-generation sequencing method (454 Life Sciences, Inc.) have increased to >250 nucleotides. These clonal read lengths make possible setting the phase of the linked polymorphisms within an exon and thus the unambiguous determination of the sequence of each HLA allele. Here we demonstrate this capacity as well as show that the throughput of the system is sufficiently high to enable a complete, 7-locus HLA class I and II typing for 24 or 48 individual DNAs in a single GS FLX sequencing run. Highly multiplexed amplicon sequencing is facilitated by the use of sample-specific internal sequence tags (multiplex identification tags or MIDs) in the primers that allow pooling of samples yet maintain the ability to assign sequences to specific individuals. We have incorporated an HLA typing software application developed by Conexio Genomics (Freemantle, Australia) that assigns HLA genotypes for these 7 loci (HLA-A, -B, -C, DRB1, DQA1, DQB1, DPB1), as well as for DRB3, DRB4, and DRB5 from 454 sequence data. The potential of this HLA sequencing system to analyze chimeric mixtures is demonstrated here by the detection of a rare HLA-B allele in a mixture of two homozygous cell lines (1/100), as well as by the detection of the rare nontransmitted maternal allele present in the blood of a severe combined immunodeficiency disease syndrome (SCIDS) patient.

The spectra of Orion A and IRC + 10216 between 72.2 and 91.1 GHz.

The complete spectra of Orion A and IRC +10216 in the range 72.2 to 91.1 GHz, obtained with the 20 m mm-wave telescope at the Onsala Space Observatory, are presented.

The detection of interstellar methylcyanoacetylene.

A new interstellar molecule, methylcyanoacetylene (CH3C3N), has been detected in the molecular cloud TMC-1. The J = 8 --> 7, J = 7 --> 6, J = 6 --> 5, and J = 5 --> 4 transitions have been observed. For the first three of these, both the K = 0 and K = 1 components are present, while for J = 5 --> 4, only the K = 0 line has been detected. The observed frequencies were calculated by assuming a value of radial velocity VLSR = 5.8 km s-1 for TMC-1, typical of other molecules in the cloud. All observed frequencies are within 10 kHz of the calculated frequencies, which are based on the 1982 laboratory constants of Moises et al., so the identification is secure. The lines are broadened by hyperfine splitting, and the J = 5 --> 4, K = 0 transition shows incipient resolution into three hyperfine components. The rotational temperature determined for these observations is quite low, with 2.7 K < or = Trot < or = 4 K. the total column density is approximately 5 x 10(12) cm-2.

Spectral scan of Orion A and IRC+10216 from 72 to 91 GHz.

The spectra of the Orion KL molecular cloud and the envelope of the carbon star IRC+10216 have been surveyed at 1 MHz resolution over the interval 72.2-91.1 GHz to a sensitivity (in terms of main beam brightness temperature) usually better than 0.1 K. Some complementary data have been obtained at selected higher and lower frequencies. Detected lines are reported in Table 1. Approximately 170 lines from 24 known interstellar molecules were detected in Orion (Table 2); the corresponding numbers for IRC+10216 are 45 and 12, respectively (Table 3). Some 15 recombination lines of hydrogen (alpha, beta, gamma, delta) and helium (alpha) are also identified in Orion (Table 2). More than 100 of the Orion lines can be attributed to astronomically new transitions, the vast majority of which belong to only five species: SO2 (in total 17 lines), CH3CH2CN (19), CH3OH (16), (CH3)2O (21) and CH3OOCH (32). The rare occurrence of unidentified lines (5 definite lines in each object) is significant, possibly indicating that, in the case of Orion, the dominant chemical constituents are recognized. For IRC+10216 our results are less conclusive in this respect, partly due to the less effective excitation conditions in the envelope. We report (or confirm) the existence in these sources of several molecules which previously had only been found elsewhere: methylformate (CH3OOCH), isocyanic acid (HNCO), cyanodiacetylene (HC5N), vinyl cyanide (CH2CHCN), ketene (H2CCO) and probably the formyl radical (HCO) in Orion; and in IRC+10216 the first species containing the methyl group, methyl cyanide (CH3CN). The first astronomical detections of the isotopically rare species 34SO2 (Orion), and 29SiS and, tentatively, Si34S (IRC+10216) are also reported, as well as a number of newly identified high energy methanol lines. Multi-transition analysis of several molecular species is presented and gives information on physical conditions and chemical abundances in the Orion KL region (Tables 5 and 6). Gaussian decomposition of 0.25 MHz resolution profiles into "ridge", "hot core" and "plateau" emissions is used in an attempt to individually characterize these subregions. Striking chemical differences among the subregions emerge: (i) The estimated abundance ratios [SO, SO2, SiO]/[CO] are enhanced by a factor approximately > 100 in the plateau relative to the ridge, [HCN, HDO]/[CO] by a factor approximately > 10, while [HCO+, OCS, HNC, HC3N]/[CO] seem to be less enhanced in the plateau (by a factor approximately >10). (ii) The ratio of chemically saturated to unsaturated species differs markedly between the hot core and the ridge cloud (specifically HC3N vs. CH3CH2CN, and CH2CHCN) with the latter region more closely resembling both the cloud TMC-1 and IRC+10216. The chemical selectivity in the ridge cloud is very pronounced, with CH3OH as abundant as H2CO while (CH3)2O and CH3OOCH are an order of magnitude less abundant. We have neither detected CH3CH2OH and CH3COOH (acetic acid)--isomers of the two latter species, respectively--nor HCOOH, CH3CHO, or CH2CHCHO. H2C2O (ketene) is two orders of magnitude less abundant than H2CO. From the isotopic species of CH3OH, OCS, and HC3N we find 12C/13C approximately 40, in agreement with independent estimates by others and a factor of two lower than the solar system isotope ratio. The observed intensities of the hydrogen recombination lines are consistent with optically thin LTE emission, indicating that our He alpha/H alpha intensity ratio is relatively model independent and thus is an accurate measure of the helium abundance. We estimate a helium abundance by mass of 28 +/- 2%. The excitation conditions and relative abundances in the IRC+10216 envelope are reviewed. It is emphasized that the low detection rate of molecular lines in this object does not necessarily imply a poorer chemistry compared with that in interstellar clouds, but is partly a reflection of low abundances and unsatisfied excitation requirements. However, the CH3CN data indicate less favorable conditions for species containing methyl groups in this environment relative to interstellar clouds. The chemistry appears to be dominated by linear, especially unsaturated carbon-chain, molecules (Table 9). Rotational temperatures for HC3N and HC5N are estimated to be on the order of 15 K. Guided by the selective chemistry in IRC+10216 we tentatively assign two close doublets at 76.2 and 98.0 GHz to the radical C3H. In addition to Orion KL and IRC+10216 a few other sources have been observed, however in a less systematic way. We present selected data for W 51 M. The W 51 M methanol data, including newly identified high energy lines, indicate a rotational temperature of about 100 K.