Aim Super high resolution-single molecule-sequence-based typing (SS-SBT) is an HLA DNA typing method for the field 4 level of allelic resolution to efficiently detect new and null alleles without ...phase ambiguity by combination of long ranged PCR amplification and next generation sequencing (NGS) technologies. We previously reported the development and application of the SS-SBT method such as long ranged PCR system from the promoter-enhancer region to 3’UTR for 11 classical HLA loci, A, B, C, DRB1, DRB3/4/5, DQA1, DQB1, DPA1 and DPB1 using NGS platforms such as Ion PGM, GS Junior and MiSeq. However, HLA alleles could not be assigned at the field 4 level in some HLA loci such as HLA-DQA1, -DPA1 and -DPB1, because SNP and indel densities to separate both of the phases were much lower than in other HLA loci. In this respect, third generation sequencers such as single molecule, real-time (SMRT®) DNA sequencer PacBio RS II (2.5∼10 kb) are expected to solve this current genotyping problem to improve the SS-SBT method. Here we report an accuracy of genotyping results obtained from SMRT® technology. Method Ten genomic DNAs (TU1∼TU10) obtained from Japanese were provided for long ranged PCR amplification using eight HLA loci specific primer pairs (A, B, C, DRB1, DQA1, DQB1, DPA1 and DPB1). Consensus sequences were obtained via HlaTools/Long Amplicon analysis such as overlapping, clustering of filtered subreads, phasing and removing PCR artifacts. Results HLA sequences obtained from 10 pooled amplicon samples applied to SMRT® technology with HlaTools/Long Amplicon analysis were compared to published reference sequences for consensus alleles. A total of 153 amplicon sequences were compared, 117 with the field 4 level sequences and 36 with the field 3 level sequences. At the field 3 level 100% of sequences were identical to reference ones while ∼70% (80/117) of sequences were identical at the field 4 level. Error sequences were mostly localized to intronic single- and di-nucleotide homopolymer regions. Conclusion The SMRT® technology is useful for highly accurate DNA typing for all the classical HLA loci.
LBP07 Ranade, Swati; Eng, Kevin; Harding, John ...
Human immunology,
04/2015, Letnik:
76, Številka:
4
Journal Article
Recenzirano
Aim The aim of this study was to evaluate PacBio sequencing technology for HLA typing of multiple DNA samples using multiplexing library preparation strategies that enable pooling of samples and ...loci. Method Three multiplexing library preparation strategies were evaluated: Multiplex sequencing using symmetric and asymmetric paired HLA amplification primers versus NGSgo® HLA amplification (GenDx) in combination with symmetric adapter ligation. Barcode-tailed HLA-locus specific amplification primers were used to amplify the full-length HLA class I gene of HLA-A, -B, and -C. For this evaluation test, 8 different 16bp barcode sequences were evaluated in symmetric and asymmetric paired multiplex sequencing. For the asymmetric paired barcodes, different barcodes occur at each end to allow for testing of 28 genomic reference DNA samples. For the symmetric paired barcodes, 8 genomic reference DNA samples were tested for the barcode-tailed amplification primers as well as the barcode-tailed adapters. Following amplification, intra-sample amplicons and loci were pooled over 3 libraries, keeping the three sequencing strategies separate for analysis. The libraries were run on the PacBio® RS II system, and the resulting sequence reads were aligned to generate a highly accurate consensus and typed using GenDx NGSengine® HLA typing software. Result We will present data on the performance of the HLA-locus specific amplification using the symmetric and asymmetric paired barcoded primer sets. We will show the effect of the amplicon pooling on typing accuracy and reliability using the different multiplexing sequencing strategies. We will show the sequencing data of equimolar pools of 3 HLA full-length genes from different heterogeneous reference samples sequenced in a single SMRT® Cell. We will also demonstrate the full-length information (all exons + introns) for allele-level genotyping along with SNP phasing information. Conclusion Using PacBio’s barcode strategy, multiple DNA samples can be sequenced for all HLA class I genes in a high-throughput setup to provide quick turn-around of HLA typing results. SMRT Sequencing and analysis using the Long Amplicon Analysis Pipeline can generate long, highly accurate DNA sequences suitable for allele-level resolution HLA typing using the NGSengine HLA typing software.
OR05 Pyo, Chul-woo; Vierra-Green, Cynthia; Pyon, Yoon Soo ...
Human immunology,
10/2014, Letnik:
75
Journal Article
Recenzirano
Aim The knowledge of complete haplotype structure and underlying genetic variation is often key in understanding gene function, phenotype, and medically relevant traits such as susceptibility to ...disease and individual response to drugs. However, genome wide SNP arrays and shotgun sequencing methods are limited in their ability to identify phased genetic variations at high resolution over extended localized regions. Methods To address this, we improved methods for targeted DNA isolation using fosmid technology and single-molecule, long-sequence-read generation that combine for complete, haplotype-resolved resequencing across extended genomic subregions. As specific applications, we targeted subregions of the human major histocompatibility complex (MHC) and the killer Ig-like receptor (KIR) locus, two of the most polymorphic genomic regions in humans. Results For the MHC region, an 800 kb segment of the MHC class II region including the highly polymorphic DR, DQ, and DP genes was targeted. Complete phased sequences were obtained encompassing the DR4 common extended haplotype – a haplotype associated with autoimmune disorders - from four class II heterozygous individuals. For the KIR locus, which spans some 200 kb including tandem repeats of highly homologous polymorphic loci, we targeted several KIR haplotype heterozygous individuals. Conclusions Complete phased sequences for all haplotypes were obtained from all individuals with a high level of data accuracy. The results demonstrate the approach is amenable to cost-effective scale-up to generate scores to hundreds of phased chromosomal sequences of extended lengths that encompass targeted genomic regions associated with disease.
The knowledge of complete haplotype structure and underlying genetic variation is often key in understanding gene function, phenotype, and medically relevant traits such as susceptibility to disease ...and individual response to drugs. However, genome wide SNP arrays and shotgun sequencing methods are limited in their ability to identify phased genetic variations at high resolution over extended localized regions.
To address this, we improved methods for targeted DNA isolation using fosmid technology and single-molecule, long-sequence-read generation that combine for complete, haplotype-resolved resequencing across extended genomic subregions. As specific applications, we targeted subregions of the human major histocompatibility complex (MHC) and the killer Ig-like receptor (KIR) locus, two of the most polymorphic genomic regions in humans.
For the MHC region, an 800kb segment of the MHC class II region including the highly polymorphic DR, DQ, and DP genes was targeted. Complete phased sequences were obtained encompassing the DR4 common extended haplotype – a haplotype associated with autoimmune disorders - from four class II heterozygous individuals. For the KIR locus, which spans some 200kb including tandem repeats of highly homologous polymorphic loci, we targeted several KIR haplotype heterozygous individuals.
Complete phased sequences for all haplotypes were obtained from all individuals with a high level of data accuracy. The results demonstrate the approach is amenable to cost-effective scale-up to generate scores to hundreds of phased chromosomal sequences of extended lengths that encompass targeted genomic regions associated with disease.
OR01 Hall, Richard J; Eng, Kevin; Hon, Lawrence ...
Human immunology,
10/2014, Letnik:
75
Journal Article
Recenzirano
Aim To implement an automated workflow for the assembly of complex immunogenetic regions using pooled fosmids and SMRT® Sequencing. Methods Here we present a simple automated workflow for the ...assembly of complete haplotypes from pools of tiled fosmids using a single library preparation and sequencing run with a quick turnaround time using an automated pipeline. The long reads generated in SMRT® Sequencing together with the hierarchical genome assembly process (HGAP) algorithm make it possible to fully assemble data with high accuracy even when the underlying structure is highly repetitive. Long reads are first preassembled into highly accurate error corrected reads before an overlap layout consensus (OLC) assembly to generate complete fosmid sequences. Fosmid sequences are trimmed of any vector before being overlapped to from a continuous haplotype. Results We show complete assembly of ∼150–200 kb haplotypes of the KIR locus, characterized by a large number of tandem repeats, from a single sequencing run. The assembly has a high base accuracy when compared to known references. Discussion The ability to haplotype and sequence complex immunogenetic regions will facilitate the integration of long-range LD in Conserved Extended Haplotypes in the MHC region and LRC region. Local fine-scale LD data will bring exciting opportunities to explore the evolution of disease associations of the immune sub-genome. A tiling of targeted fosmids can be used to clone extended lengths of genomic DNA, 100s of kb in length, but repeat complexity in regions of particular interest, such as the major histocompatibility complex (MHC) and the killer Ig-like receptor (KIR) locus, means that sequence assembly of complete haplotypes is difficult and often requires expert knowledge. This simple and robust assembly approach can be scaled-up allowing a complex genomic region to be sequenced in a population genetics setup. Such sequencing would be of high value in disease association research. R.J. Hall: Employee; Company/Organization; Pacific Biosciences. K. Eng: Employee; Company/Organization; Pacific Biosciences. L. Hon: Employee; Company/Organization; Pacific Biosciences. D.E. Geraghty: Employee; Company/Organization; Scisco Genetics Inc. S. Ranade: Employee; Company/Organization; Pacific Biosciences.
To sequence entire HLA class I genes of 45 DNA samples in a single sequencing reaction using DNA-barcode-labelled primers and Single Molecule, Real-Time (SMRT®) DNA sequencing technology.
Our ...previous experience of sequencing full-length HLA class I genes using SMRT® technology has shown that it has read length capabilities and accuracy amenable for definitive allele-level resolution HLA typing. Further experiments to determine multiplexing using DNA barcodes have shown that multiplexing up to 20 DNA samples for a single HLA locus or 8 DNA samples for multiple loci, is possible. Forty-five DNA samples were selected for HLA class I typing and were d derived from different DNA sources (blood, saliva and umbilical cord blood). Nine of these DNA samples had previously been identified as having novel alleles at one of their HLA class I loci using standard HLA typing methods. Whole gene amplicons were generated for the HLA class I genes. Each of the forty-five DNA samples were amplified for HLA-A, -B and -C using generic primers tagged with a DNA barcode unique to that sample resulting in 135 barcoded amplicons.
Sufficient numbers of reads were obtained for each locus of each sample to enable HLA allele calling (mean 145; range 47–385) with mean a QV>75. 252 HLA class I alleles were expected from the 45 DNA samples when homozygous loci were considered. The HLA types of 219 of these alleles immediately matched the types observed previously. The consensus sequences also confirmed the presence and phasing of the variants within the nine novel alleles. The consensus sequences on the 24 remaining alleles contained novel differences when compared to existing reference sequences in the IMGT/HLA Database. Sanger sequencing is being used to verify these novel positions.
SMRT DNA sequencing has the capability and the accuracy to facilitate definitive allele-level resolution HLA typing. We have shown for the first time that it is possible to use this method to sequence multiple DNA samples for multiple HLA class I genes using DNA barcode technology, to a level that makes it viable for routine use as a high-throughput methodology. The identification of previously unknown mismatches between recipients and their donors could significantly improve stem cell transplant outcome.
S. Ranade:Employee; Company/Organization; Pacific Biosciences. K. Eng:Employee; Company/Organization; Pacific Biosciences. B. Bowman:Employee; Company/Organization; Pacific Biosciences. L. Hepler:Employee; Company/Organization; Pacific Biosciences.
To sequence the full length of HLA Class I genes using single molecule real-time (SMRT®) sequencing technology.
Two samples were amplified by Long-Range PCR at HLA-A, B, C loci. Full lengths of these ...genes (∼3.2kb) were generated with custom designed, barcoded primers targeting 5’UTR and 3’UTR. DNA libraries were prepared by ligating SMRTbell™ adaptors to the HLA amplicons. This produced closed-circular structures consisting of a double-stranded insert (full length HLA amplicon), and a single-stranded hairpin loop (SMRTbell™) on either end. Sequencing primer was annealed to the open/single-stranded portion of the hairpin adaptor. Streptavidin-bound DNA polymerase was added to the primer-template complex enabling the binding of the template to the bottom of nano-sized wells on the SMRT Cell. DNA synthesis of fluorescently labeled nucleotides was observed in real-time on the PacBio® RS instrument. The pooled libraries were run on 6 SMRT Cells using either 45 or 90 minute data collection time per SMRT Cell. Individual sequence reads ⩾1,000 bases were parsed by sample-specific barcode sequence and were then aligned using BWA-SW. Alignment data were analyzed with SAM Tools and custom written code.
Sequence and Alignment Statistics: Table 1.
We demonstrate real-time sequencing of HLA genes from single DNA molecules. This technology offers great potential for HLA genotyping and further characterization of the MHC due to the length and phase of sequences obtained, speed, and ease of sample prep.
Ranade: Pacific Biosciences Inc.: Employee. Sethuraman: Pacific Biosciences Inc.: Employee. Chin: Pacific Biosciences Inc.: Employee. Robinson: Pacific Biosciences Inc.: Employee.
Aim To sequence the full length of HLA Class I genes using single molecule real-time (SMRT® ) sequencing technology. Methods Two samples were amplified by Long-Range PCR at HLA-A, B, C loci. Full ...lengths of these genes (∼3.2 kb) were generated with custom designed, barcoded primers targeting 5’UTR and 3’UTR. DNA libraries were prepared by ligating SMRTbell™ adaptors to the HLA amplicons. This produced closed-circular structures consisting of a double-stranded insert (full length HLA amplicon), and a single-stranded hairpin loop (SMRTbell™) on either end. Sequencing primer was annealed to the open/single-stranded portion of the hairpin adaptor. Streptavidin-bound DNA polymerase was added to the primer-template complex enabling the binding of the template to the bottom of nano-sized wells on the SMRT Cell. DNA synthesis of fluorescently labeled nucleotides was observed in real-time on the PacBio® RS instrument. The pooled libraries were run on 6 SMRT Cells using either 45 or 90 minute data collection time per SMRT Cell. Individual sequence reads ⩾1,000 bases were parsed by sample-specific barcode sequence and were then aligned using BWA-SW. Alignment data were analyzed with SAM Tools and custom written code. Results Sequence and Alignment Statistics: Table 1 . Conclusions We demonstrate real-time sequencing of HLA genes from single DNA molecules. This technology offers great potential for HLA genotyping and further characterization of the MHC due to the length and phase of sequences obtained, speed, and ease of sample prep. Ranade: Pacific Biosciences Inc.: Employee. Sethuraman: Pacific Biosciences Inc.: Employee. Chin: Pacific Biosciences Inc.: Employee. Robinson: Pacific Biosciences Inc.: Employee.
The development of the
Drosophila visual system utilizes two members of the highly conserved Six-Homeobox family of transcription factor, Sine oculis and Optix. Although in vitro studies have ...detected differences in DNA-binding and interactions with some co-factors, questions remain as to what extent the activity for these two transcriptional regulators is redundant or specific in vivo. In this work, we show that the So
D mutation within the Six domain does not abolish DNA–protein interactions, but alters co-factor binding specificity to resemble that of Optix. A mutation in the same region of Optix alters its activity in vivo. We propose that the dominant mutant phenotype is primarily due to an alteration in binding properties of the Sine oculis protein and that distinct partner interactions is one important mechanism in determining significant functional differences between these highly conserved factors during eye development.
Aim To sequence entire HLA class I genes of 45 DNA samples in a single sequencing reaction using DNA-barcode-labelled primers and Single Molecule, Real-Time (SMRT®) DNA sequencing technology. Method ...Our previous experience of sequencing full-length HLA class I genes using SMRT® technology has shown that it has read length capabilities and accuracy amenable for definitive allele-level resolution HLA typing. Further experiments to determine multiplexing using DNA barcodes have shown that multiplexing up to 20 DNA samples for a single HLA locus or 8 DNA samples for multiple loci, is possible. Forty-five DNA samples were selected for HLA class I typing and were d derived from different DNA sources (blood, saliva and umbilical cord blood). Nine of these DNA samples had previously been identified as having novel alleles at one of their HLA class I loci using standard HLA typing methods. Whole gene amplicons were generated for the HLA class I genes. Each of the forty-five DNA samples were amplified for HLA-A, -B and -C using generic primers tagged with a DNA barcode unique to that sample resulting in 135 barcoded amplicons. Results Sufficient numbers of reads were obtained for each locus of each sample to enable HLA allele calling (mean 145; range 47–385) with mean a QV > 75. 252 HLA class I alleles were expected from the 45 DNA samples when homozygous loci were considered. The HLA types of 219 of these alleles immediately matched the types observed previously. The consensus sequences also confirmed the presence and phasing of the variants within the nine novel alleles. The consensus sequences on the 24 remaining alleles contained novel differences when compared to existing reference sequences in the IMGT/HLA Database. Sanger sequencing is being used to verify these novel positions. Conclusion SMRT DNA sequencing has the capability and the accuracy to facilitate definitive allele-level resolution HLA typing. We have shown for the first time that it is possible to use this method to sequence multiple DNA samples for multiple HLA class I genes using DNA barcode technology, to a level that makes it viable for routine use as a high-throughput methodology. The identification of previously unknown mismatches between recipients and their donors could significantly improve stem cell transplant outcome. S. Ranade: Employee; Company/Organization; Pacific Biosciences. K. Eng: Employee; Company/Organization; Pacific Biosciences. B. Bowman: Employee; Company/Organization; Pacific Biosciences. L. Hepler: Employee; Company/Organization; Pacific Biosciences.
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