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Aim The Roche GS GType Assay is an amplicon based NGS approach for high-throughput, high-resolution HLA genotyping. We evaluated the ability of this assay together with Conexio Assign MPS software to define high resolution HLA alleles by sequencing 59 samples and examining the concordance between prior typing information and the GType Assay. Our goal was to determine whether this single method would significantly reduce ambiguity in the assignment of HLA class I and II alleles. Methods PCR amplification was performed using the Very High Resolution HLA GType Assay which consists of three 96-well plates (MR, HR and VHR) containing dried-down exon-specific primers. The MR plate contains 8 primer pairs amplifying exons 2 and 3 of HLA-A, -B, and -C, and exon 2 of DRB1 (plus DRB3, 4, 5) and DQB1. The HR plate contains 6 primer pairs amplifying exon 4 of HLA-A, -B, and -C, exon 2 of DPB1 and DQA1, and exon 3 of DQB1. The VHR plate amplifies 8 exons covering exons 1 and 5 of HLA-A, -B, -C, exon 6 and 7 of HLA-C, exon 2 of DPA1, and exon 3 of DRB1 (plus DRB3, 4, 5). Using 11 unique multiplex identifiers (MIDs) allows pooling 5 samples with 22 amplicons and a negative control on one GS Jr. run. Resulting FNA files were analyzed by Conexio software. Results Sequence analysis of 59 individual samples (55 bone marrow donors/recipients, 1 lung recipient, and 3 controls) including 18 rare alleles demonstrated 100% concordance with genotypes previously high resolution typed by a combination of Sanger sequencing, sequence-specific-primer (SSP), and sequence-specific-oligonucleotide probe (SSOP) methodologies. Increased sequence coverage allowed us to detect additional variants (silent substitutions) providing third field genotyping information. Some residual genotype ambiguity was present, largely due to incomplete exon coverage or incomplete reference sequence in the IMGT/HLA database. No additional methodology was required to obtain the high resolution allele. Conclusions Our results provide support for the use of amplicon based high-throughput sequencing for high-resolution HLA genotyping by NGS. The technique is accurate, and results in reduced genotype ambiguity when compared to standard Sanger sequencing; however, further primer design development and genotyping software are necessary before application for clinical use. [ABSTRACT FROM AUTHOR]

Aim: The clonal property of next generation sequencing systems allows for the quantitative analysis of mixed samples by simply counting sequences corresponding to the component alleles. Previously, we developed a method using 454 amplicon sequencing and Conexio software for high resolution and high throughput genotyping of the HLA class I and class II loci. This system was used to analyze the blood of a SCID child and estimate the proportion of maternal cells by counting HLA-C allelic sequence reads corresponding to the non-transmitted maternal allele. Here we report the development of a system that allows us to quantify HLA allelic mixtures in plasma. Methods: DNA from plasma or contrived mixtures of cell lines was amplified using primers that targeted a short region (∼150 bp to amplify small DNA fragments in plasma) of HLA DPB1 or DQB1 exon 2. Amplicons were further amplified by emulsion PCR and sequenced on a 454 GS FLX or GS Junior. Sequences were examined using modified Conexio Assign ATF 454 software. This software allowed for rapid digital analysis of each DPB1 or DQB1 allele. We also analyzed mixtures using the Illumina MiSeq. Results: Using the modified Conexio software, minority HLA alleles in a mixture were readily identified and separated from background “noise” i.e., sequences generated by PCR or sequencing errors. In mixtures of two heterozygous cell lines, the minority HLA alleles could be detected at 0.5% with 1 ng (∼140 diploid genomes) DNA input. In 3/3 plasma samples from kidney transplant recipients undergoing acute or chronic rejection, donor HLA alleles were detected at an average of 0.6% of the total of donor plus recipient alleles. Conclusions: We have developed a system that allows the sensitive and precise analysis of mixtures using either 454 or Illumina MiSeq sequencing technology. This method could be useful in numerous clinical applications; we have demonstrated the detection of donor HLA alleles in the plasma of kidney transplant recipients undergoing rejection. Holcomb: Roche: Employee. Rastrou: Roche: Employee. Hoglund: Roche: Employee. Goodridge: Conexio Genomics: Employee. Erlich: Roche: Employee. [Copyright &y& Elsevier]

Aim: Genotyping of DRB1 and DRB3/4/5 is performed in our lab using generic DRB PCR primers, followed by sequencing on the 454 GS FLX and use of Conexio Genomics software. During genotyping homozygous cell lines, we detected additional sequences at about 7% of the read depth of the reported true allele. These artifacts appeared to be PCR crossovers between the DRB1 and DRB3/4/5 sequences. The crossovers usually had one or more mismatches with the IMGT database but occasionally matched a very rare allele (e.g. DRB1∗03:42). We performed a study to determine if these sequences were in vitro artifacts and to determine if some rare DRB alleles in the IMGT database might, in fact, be in vitro artifacts that had been submitted. Methods: To test for in vitro artifact formation during late cycles of genomic PCR, we amplified the DRB loci from 21 homozygous cells lines under both our standard PCR conditions (35 cycles) and a reduced number of cycles (28), followed by 454 sequencing. Using the same PCR conditions, we also sequenced the DRB loci of four samples, each of which had previously been identified as having a novel DRB allele that was subsequently submitted to the IMGT database. Results: For all homozygous 21 cell lines, amplification for 35 cycles gave low abundance sequences as possible “second alleles.” In 36% of the cases, these artifactual sequences corresponded to named alleles. Such sequences were not detected with amplification for 28 cycles. In the case of 4 samples with rare alleles previously submitted to the IMGT, we found that 3 were true alleles. The fourth, while not a crossover product, contained a sequencing error which was revealed by our 454 HLA genotyping assay. Conclusions: Formation of in vitro crossover products of the DRB loci can occur in late cycles of PCR. PCR conditions have been identified that minimize generation of these in vitro artifacts. Clonal sequencing on the 454 GS FLX is a valuable method for revealing both these artifacts and traditional sequencing errors. [Copyright &y& Elsevier]

Aim: We have previously reported the development of very high resolution (VHR) HLA genotyping, designed to resolve the majority of both the “common and well documented alleles” and the null alleles using the 454 GS FLX Sequencing System and Conexio genotyping software. Amplicon library construction was performed using 22 PCR fusion primers, containing genome target specific sequence with 454 sequencing adaptors. The VHR assay performed well but required extensive handling of individual amplicons. To simplify the workflow, we prepared the amplicon libraries by three alternative procedures. Methods: PCR amplicons were generated from 10 cell lines for: HLA-A/B exons 1-5, HLA-C exons 1-7 and some intronic class I regions; DPA1, DPB1, DQA1 exon 2; and DQB1, DRB exons 2 and 3. Amplification required 22 pairs of fusion primers (14 primer pairs in the GS GType HLA primers plates from 454, plus 8 amplicons in a third plate) with 10 Multiplex Identifiers (MIDs), or 22 primer pairs each containing, at the 5′end, a universal sequence which matched the 3′ end of a set of primers that contained 10 MIDs and the 454 sequencing adaptors. Amplicons derived from fusion primers were purified, quantified, diluted to a standard concentration, then pooled OR pooled prior to the other operations. Amplicons generated by the four primer system on the Fluidigm Access Array™ were handled by the latter procedure. Pools of amplicons were sequenced on the GS FLX and genotyped with Conexio software. Results: All three procedures resulted in comparable genotyping (concordance up to >98%). Pooling amplicons immediately following the genomic PCR gave a reduction in the number of pipetting steps necessary to produce an amplicon library by ∼80% and ∼90% using fusion primers and the four primer system, respectively. Conclusions: Pooling amplicons early in the 454 library preparation procedure greatly simplifies the workflow of the 22 primer VHR HLA genotyping assay for fusion primers as well as the 4 primer system. Holcomb: Roche: Employee. Hoglund: Roche: Employee. Williams: Roche: Grant Research. Erlich: Roche: Employee. [Copyright &y& Elsevier]

Aim The circulation of kidney transplant recipients has been reported to contain DNA from the graft, associated with rejection. We previously reported the development of a sensitive, accurate and precise method to quantitatively analyze contrived mixtures of two cell-line DNAs by next generation sequencing (NGS) of short HLA amplicons. Here we report the application of this method to analyze the cell-free circulating (cfc) DNA from kidney transplant recipients that fell into one of four categories at that time of sample collection: (1) successful transplant (Tx), (2) experiencing acute rejection (AR), (3) experiencing chronic rejection (CR), or (4) experiencing acute dysfunction (ADNR) but no rejection. Methods DNA from recipient plasma was amplified using primers that targeted a short region of HLA-DPB1 or DQB1 exon 2 (⩽150 bp to amplify small DNA fragments in plasma). Primers included Illumina Miseq tags. A second round of PCR was performed to introduce Miseq indices (to identify individual samples) and sequencing adapters. Amplicons were sequenced on a 2 × 150 output run to obtain sequence in both directions and analyzed with a modified version of Conexio Assign ATF 454 software as well as SWISSA, an in-house NGS software. Both softwares allowed for rapid digital analysis of HLA alleles present from graft recipients and donors. Results Donor HLA alleles in recipient plasma were readily identified and separated from background “noise” i.e., sequences generated by PCR or sequencing errors. The average number of sequence reads when processing with SWISSA was 146,000 per sample. Using cell-line mixtures, with an input of 8 ng of DNA, we detected alleles down to 0.25% (the lowest amount we tested). In a study of 29 patients, the average percentages of donor DNA were 2.1 ± 2.3, 1.3 ± 1.4, 0.5 ± 0.4 and 0.2 ± 0.3 for patients who had AR, CR, ADNR or Tx, respectively. Conclusion Illumina MiSeq sequencing can be used to noninvasively detect and quantify the presence of donor DNA in kidney transplant recipients by analysis of plasma. The average percentage of cfc donor DNA correlated with an increasing severity of rejection in this initial exploratory study. [ABSTRACT FROM AUTHOR]

Aim: Next generation sequencing of HLA exonic amplicons with the 454 Life Sciences GS FLX System and Conexio Assign TM -ATF software provides high resolution, high throughput HLA genotyping for 8 class I and class II loci (Bentley et al., 20091, Holcomb et al., 20112). HLA typing of potential donors for Unrelated Bone Marrow Donor Registries requires using a subset of these loci at high sample throughput and low cost per sample. To maximize the throughput, we have incorporated the Fluidigm® Access Array TM system to simplify amplicon library preparation and allow the efficient introduction of MIDs (Multiplex Identifiers) or barcodes. Methods: For this application, a streamlined amplicon sequencing workflow employing many different Multiplex Identifiers (MIDs) enables multiplexed sequencing of a large number of samples, allowing researchers to meet these cost targets. The Fluidigm® 48 x 48 Access Array TM system and the “4 primer” approach provides an efficient way to achieve parallel semi-automated genomic PCRs with simultaneous incorporation of 48 different MIDs corresponding to 48 genomic DNA samples, and up to 48 different primer pairs, making possible 2,304 reactions in one amplification run. Minimal volumes (calculated to be about 45% less cost per run) of reagents are used in this micro- fluidics-based system. During genomic PCR, the outer set of primers containing the MIDs and the 454 adaptor sequences are incorporated into the amplicons generated by the inner set of HLA specific primers containing a complementary “universal” Fluidigm tag. Pools of the resulting amplicons are used for emulsion PCR and clonal sequencing on the 454 GS FLX System, as well as genotyping with Conexio Assign TM -ATF software. Results: Using the Access Array system, we have successfully (100% concordance with known genotypes) genotyped 192 samples with 8 primer pairs (covering exons 2 and 3 in HLA-A, B, C and Exon 2 in DRB1, DRB3/4/5 and DQB1) using 96 MIDs per region in a single GS FLX run on a 2 region PicoTiterPlate™ (PTP) and 96 samples using 48 MIDs per region in a 4 region PTP. An average of 166 and 137 sequence reads per amplicon were recovered respectively. We have also genotyped, in a single run, 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 175 sequence reads per amplicon at 100% concordance. The system reduces the overall time for the entire process (192 samples) from 8 days for manual processing to about 4 days with 1 FTE. [Copyright &y& Elsevier]