Seqanswers Leaderboard Ad

Collapse

Announcement

Collapse
No announcement yet.
X
 
  • Filter
  • Time
  • Show
Clear All
new posts

  • PubMed: Ultra-high resolution HLA genotyping and allele discovery by highly multiplex

    Syndicated from PubMed RSS Feeds

    Ultra-high resolution HLA genotyping and allele discovery by highly multiplexed cDNA amplicon pyrosequencing.

    BMC Genomics. 2012 Aug 6;13(1):378

    Authors: Lank SM, Golbach BA, Creager HM, Wiseman RW, Keskin DB, Reinherz EL, Brusic V, O'Connor DH

    Abstract
    ABSTRACT: BACKGROUND: High-resolution HLA genotyping is a critical diagnostic and research assay. Current methods rarely achieve unambiguous high-resolution typing without making population-specific frequency inferences due to a lack of locus coverage and difficulty in exon-phase matching. Achieving high-resolution typing is also becoming more challenging with traditional methods as the database of known HLA alleles increases. RESULTS: We designed a cDNA amplicon-based pyrosequencing method to capture 94% of the HLA class I open-reading-frame with only two amplicons per sample, and an analogous method for class II HLA genes, with a primary focus on sequencing the DRB loci. We present a novel Galaxy server-based analysis workflow for determining genotype. During assay validation, we performed two GS Junior sequencing runs to determine the accuracy of the HLA class I amplicons and DRB amplicon at different levels of multiplexing. When 116 amplicons were multiplexed, we unambiguously resolved 99%of class I alleles to four- or six-digit resolution, as well as 100% unambiguous DRB calls. The second experiment, with 271 multiplexed amplicons, missed some alleles, but generated high-resolution, concordant typing for 93% of class I alleles, and 96% for DRB1 alleles. In a third, preliminary experiment we attempted to sequence novel amplicons for other class II loci with mixed success. CONCLUSIONS: The presented assay is higher-throughput and higher-resolution than existing HLA genotyping methods, and suitable for allele discovery or large cohort sampling. The validated class I and DRB primers successfully generated unambiguously high-resolution genotypes, while further work is needed to validate additional class II genotyping amplicons.


    PMID: 22866951 [PubMed - as supplied by publisher]



    More...

Latest Articles

Collapse

  • seqadmin
    Strategies for Sequencing Challenging Samples
    by seqadmin


    Despite advancements in sequencing platforms and related sample preparation technologies, certain sample types continue to present significant challenges that can compromise sequencing results. Pedro Echave, Senior Manager of the Global Business Segment at Revvity, explained that the success of a sequencing experiment ultimately depends on the amount and integrity of the nucleic acid template (RNA or DNA) obtained from a sample. “The better the quality of the nucleic acid isolated...
    03-22-2024, 06:39 AM
  • seqadmin
    Techniques and Challenges in Conservation Genomics
    by seqadmin



    The field of conservation genomics centers on applying genomics technologies in support of conservation efforts and the preservation of biodiversity. This article features interviews with two researchers who showcase their innovative work and highlight the current state and future of conservation genomics.

    Avian Conservation
    Matthew DeSaix, a recent doctoral graduate from Kristen Ruegg’s lab at The University of Colorado, shared that most of his research...
    03-08-2024, 10:41 AM

ad_right_rmr

Collapse

News

Collapse

Topics Statistics Last Post
Started by seqadmin, Yesterday, 06:37 PM
0 responses
11 views
0 likes
Last Post seqadmin  
Started by seqadmin, Yesterday, 06:07 PM
0 responses
10 views
0 likes
Last Post seqadmin  
Started by seqadmin, 03-22-2024, 10:03 AM
0 responses
51 views
0 likes
Last Post seqadmin  
Started by seqadmin, 03-21-2024, 07:32 AM
0 responses
67 views
0 likes
Last Post seqadmin  
Working...
X