166-P Want a good read? Next-generation HLA sequencing using the Roche 454 GS FLX platform for five NIH projects

Next-Generation HLA Sequencing Using the 454 GS FLX System

“…In an international alpha-trial testing of 20 blinded samples, the 454 Life Sciences GS FLX System sequencing method demonstrated 98 % concordance with known genotypes between eight participating labs [2]. In our other studies, using at least one control per PTP region per run, 100 % of control sample genotypes (previously characterized by Sanger Sequencing with ambiguity reduction) were concordant [4, 5]. The average rate of detection of novel alleles was ~0.04 % (8/19,069 loci) in Caucasoid study populations [4, 5].…”

“…In our other studies, using at least one control per PTP region per run, 100 % of control sample genotypes (previously characterized by Sanger Sequencing with ambiguity reduction) were concordant [4, 5]. The average rate of detection of novel alleles was ~0.04 % (8/19,069 loci) in Caucasoid study populations [4, 5]. …”

“… For a given locus, the percentage of genotype calls that are unambiguous at the two field level (previously known as “four digit level”) (labeled 1) versus those that are composed of an ambiguity string of two through five or more genotype calls (labeled 2 through 5+) using the 454/Roche Applied Science HLA primers and GS FLX sequencing system in a study on 1,000 Caucasians [4, 5]. (a) Call percentages when expression variants are considered (as would be required for hematopoietic stem cell transplantation).…”

“…Table 2 illustrates the fraction of unambiguous genotype calls and of genotype calls that consist of ambiguity strings of varying lengths observed using 454/Roche Applied Science primers for sequencing class I (exons 2–4) and class II DQB1 (exon 2 and 3) and DRB1, DQA1, DPB1 (exon 2) in a study of 1,000 samples [4, 5]. Note that non-Caucasoid populations would be expected to have fewer ambiguous genotypes, due to unique HLA sequences in these regions.…”

“…The latter changes are useful for more robust generation of long amplicons. The DRB crossover products are a minor in vitro PCR artifact that is revealed by NGS; the ability to detect this readily identifiable population of sequences increases with clonal sequencing because we can detect very minor sequence populations [5]. …”