Allelotyping by massively parallel pyrosequencing of SNP-carrying trinucleotide threads.
Here we present an approach for allelotyping combining the multiplexing features of the trinucleotide threading (TnT) method with pooling of genomic DNA and massively parallel pyrosequencing, enabling reliable allele frequency estimation in large cohorts. The approach offers several benefits as compared to array-based methods and allows undertaking highly complex studies without compromising accuracy, while keeping the workload to a minimum. This proof-of-concept study involves formation of trinucleotide threads, targeting a total of 147 single-nucleotide polymorphisms (SNPs) related to obesity and cancer, for multiplex amplification and allele extraction from a pool of 462 genomes, followed by massively parallel pyrosequencing. Approximately 177k reads were approved, identified, and assigned to SNP-carrying threads rendering representative allele frequencies in the cohort.
Pettersson
Erik
E
Department of Gene Technology, Royal Institute of Technology (Kungliga Tekniska Högskolan, KTH), AlbaNova University Center, Stockholm, Sweden.
Zajac
Pawel
P
Ståhl
Patrik L
PL
Jacobsson
Josefin A
JA
Fredriksson
Robert
R
Marcus
Claude
C
Schiöth
Helgi B
HB
Lundeberg
Joakim
J
Ahmadian
Afshin
A
D000483:Alleles; D005787:Gene Frequency; D006801:Humans; D009711:Nucleotides / Q000378:metabolism*; D020641:Polymorphism, Single Nucleotide / Q000235:genetics*; D017422:Sequence Analysis, DNA / Q000379:methods*
D009711:Nucleotides