Computerized polymorphic marker identification: experimental validation and a predicted human polymorphism catalog

Abstract

A computational system for the prediction of polymorphic loci directly and efficiently from human genomic sequence was developed and verified. A suite of programs, collectively called POMPOUS (polymorphic marker prediction of ubiquitous simple sequences) detects tandem repeats ranging from dinucleotides up to 250 mers, scores them according to predicted level of polymorphism, and designs appropriate flanking primers for PCR amplification. This approach was validated on an approximately 750-kilobase region of human chromosome 3p21.3, involved in lung and breast carcinoma homozygous deletions. Target DNA from 36 paired B lymphoblastoid and lung cancer lines was amplified and allelotyped for 33 loci predicted by POMPOUS to be variable in repeat size. We found that among those 36 predominately Caucasian individuals 22 of the 33 (67%) predicted loci were polymorphic with an average heterozygosity of 0.42. Allele loss in this region was found in 27/36 (75%) of the tumor lines using these markers. POMPOUS provides the genetic researcher with an additional tool for the rapid and efficient identification of polymorphic markers, and through a World Wide Web site, investigators can use POMPOUS to identify polymorphic markers for their research. A catalog of 13,261 potential polymorphic markers and associated primer sets has been created from the analysis of 141,779,504 base pairs of human genomic sequence in GenBank. This data is available on our Web site (pompous.swmed.edu) and will be updated periodically as GenBank is expanded and algorithm accuracy is improved.

Figure 1

Program flow. pompous takes as input a sequence of length up to 250,000 bases in fasta format and processes it first by a parallel sequence homology search (sppblastn), which then is parsed by tandmin for hits with the properties listed in Table 1. The sequence then is scanned for larger tandem repeats by tandmax, and the outputs are consolidated. pompous then converts the consolidated data into a format that is usable by primo (regions file), and primo uses this file to predict flanking oligonucleotide primers for subsequent PCR amplification.

Figure 2

Representative allelotyping results. Polyacrylamide allelotyping gels for paired B lymphoblastoid and tumor DNAs visualized by autoradiography. (A) Marker LUCA2.2, (ca)_n repeat. (B) LUCA20.2, (aaag)_n(aagg)_n repeat. The bracket above each lane contains DNA pairs from the same individual, first normal DNA and second tumor DNA. Arrows denote different alleles for each marker. DEL, H1450 deletion line; HET, retention of heterozygosity; LOH, loss of heterozygosity; NI, noninformative.