Lauris Kaplinski

MultiPLX: automatic grouping and evaluation of PCR primers

UNLABELLED MultiPLX is a new program for automatic grouping of PCR primers. It can use many different parameters to estimate the compatibility of primers, such as primer-primer interactions, primer-product interactions, difference in melting temperatures, difference in product length and the risk of generating alternative products from the template. A unique feature of the MultiPLX is the ability to perform automatic grouping of large number (thousands) of primer pairs. AVAILABILITY Binaries for Windows, Linux and Solaris are available from http://bioinfo.ebc.ee/download/. A graphical version with limited capabilities can be used through a web interface at http://bioinfo.ebc.ee/multiplx/. The source code of the program is available on request for academic users. CONTACT maido.remm@ut.ee.

Label-free, multiplexed detection of bacterial tmRNA using silicon photonic microring resonators

A label-free biosensing method for the sensitive detection and identification of bacterial transfer-messenger RNA (tmRNA) is presented employing arrays of silicon photonic microring resonators. Species specific tmRNA molecules are targeted by complementary DNA capture probes that are covalently attached to the sensor surface. Specific hybridization is monitored in near real-time by observing the resonance wavelength shift of each individual microring. The sensitivity of the biosensing platform allowed for detection down to 53 fmol of Streptococcus pneumoniae tmRNA, equivalent to approximately 3.16×10(7) CFU of bacteria. The simplicity and scalability of this biosensing approach makes it a promising tool for the rapid identification of different bacteria via tmRNA profiling.

GENOMEMASKER package for designing unique genomic PCR primers

BackgroundThe design of oligonucleotides and PCR primers for studying large genomes is complicated by the redundancy of sequences. The eukaryotic genomes are particularly difficult to study due to abundant repeats. The speed of most existing primer evaluation programs is not sufficient for large-scale experiments.ResultsIn order to improve the efficiency and success rate of automatic primer/oligo design, we created a novel method which allows rapid masking of repeats in large sequence files, for example in eukaryotic genomes. It also allows the detection of all alternative binding sites of PCR primers and the prediction of PCR products. The new method was implemented in a collection of efficient programs, the GENOMEMASKER package. The performance of the programs was compared to other similar programs. We also modified the PRIMER3 program, to be able to design primers from lowercase-masked sequences.ConclusionThe GENOMEMASKER package is able to mask the entire human genome for non-unique primers within 6 hours and find locations of all binding sites for 10 000 designed primer pairs within 10 minutes. Additionally, it predicts all alternative PCR products from large genomes for given primer pairs.

The whole genome tagSNP selection and transferability among HapMap populations.

One of the crucial issues of association studies is the selection of markers. One possible approach would be to select tagging SNPs (tSNPs) according the HapMap information. In this study we present the number of tSNPs required for the association analysis of the entire human genome for all available HapMap population samples: CEPH, Nigerian, Chinese and Japanese. For future association studies, it is also important to know how well the tSNP set of one population sample can describe the markers of another population. Therefore, we have calculated the proportion of markers adequately described by tSNPs and how many additional tSNPs we need to describe all markers of another population.

MultiPLX: automatic grouping and evaluation of PCR primers.

In this chapter, we describe MultiPLX-a tool for automatic grouping of PCR primers for multiplexed PCR. Both generic working principle and step-by-step practical procedures with examples are presented. MultiPLX performs grouping by calculating many important interaction levels between the different primer pairs and then distributes primer pairs to groups, so that the strength of unwanted interactions is kept below user-defined compatibility level. In addition, it can be used to select optimal primer pairs for multiplexing from list of candidates. MultiPLX can be downloaded from http://bioinfo.ut.ee/download/. Graphical Web-based interface to most functions of MultiPLX is available at http://bioinfo.ut.ee/multiplx/.

Detection of NASBA amplified bacterial tmRNA molecules on SLICSel designed microarray probes

BackgroundWe present a comprehensive technological solution for bacterial diagnostics using tmRNA as a marker molecule. A robust probe design algorithm for microbial detection microarray is implemented. The probes were evaluated for specificity and, combined with NASBA (Nucleic Acid Sequence Based Amplification) amplification, for sensitivity.ResultsWe developed a new web-based program SLICSel for the design of hybridization probes, based on nearest-neighbor thermodynamic modeling. A SLICSel minimum binding energy difference criterion of 4 kcal/mol was sufficient to design of Streptococcus pneumoniae tmRNA specific microarray probes. With lower binding energy difference criteria, additional hybridization specificity tests on the microarray were needed to eliminate non-specific probes. Using SLICSel designed microarray probes and NASBA we were able to detect S. pneumoniae tmRNA from a series of total RNA dilutions equivalent to the RNA content of 0.1-10 CFU.ConclusionsThe described technological solution and both its separate components SLICSel and NASBA-microarray technology independently are applicative for many different areas of microbial diagnostics.

GenomeTester4: a toolkit for performing basic set operations - union, intersection and complement on k-mer lists

BackgroundK-mer-based methods of genome analysis have attracted great interest because they do not require genome assembly and can be performed directly on sequencing reads. Many analysis tasks require one to compare k-mer lists from different sequences to find words that are either unique to a specific sequence or common to many sequences. However, no stand-alone k-mer analysis tool currently allows one to perform these algebraic set operations.FindingsWe have developed the GenomeTester4 toolkit, which contains a novel tool GListCompare for performing union, intersection and complement (difference) set operations on k-mer lists. We provide examples of how these general operations can be combined to solve a variety of biological analysis tasks.ConclusionsGenomeTester4 can be used to simplify k-mer list manipulation for many biological analysis tasks.

FastGT: an alignment-free method for calling common SNVs directly from raw sequencing reads

We have developed a computational method that counts the frequencies of unique k-mers in FASTQ-formatted genome data and uses this information to infer the genotypes of known variants. FastGT can detect the variants in a 30x genome in less than 1 hour using ordinary low-cost server hardware. The overall concordance with the genotypes of two Illumina “Platinum” genomes is 99.96%, and the concordance with the genotypes of the Illumina HumanOmniExpress is 99.82%. Our method provides k-mer database that can be used for the simultaneous genotyping of approximately 30 million single nucleotide variants (SNVs), including >23,000 SNVs from Y chromosome. The source code of FastGT software is available at GitHub (https://github.com/bioinfo-ut/GenomeTester4/).

Haplotype Phasing and Inheritance of Copy Number Variants in Nuclear Families

DNA copy number variants (CNVs) that alter the copy number of a particular DNA segment in the genome play an important role in human phenotypic variability and disease susceptibility. A number of CNVs overlapping with genes have been shown to confer risk to a variety of human diseases thus highlighting the relevance of addressing the variability of CNVs at a higher resolution. So far, it has not been possible to deterministically infer the allelic composition of different haplotypes present within the CNV regions. We have developed a novel computational method, called PiCNV, which enables to resolve the haplotype sequence composition within CNV regions in nuclear families based on SNP genotyping microarray data. The algorithm allows to i) phase normal and CNV-carrying haplotypes in the copy number variable regions, ii) resolve the allelic copies of rearranged DNA sequence within the haplotypes and iii) infer the heritability of identified haplotypes in trios or larger nuclear families. To our knowledge this is the first program available that can deterministically phase null, mono-, di-, tri- and tetraploid genotypes in CNV loci. We applied our method to study the composition and inheritance of haplotypes in CNV regions of 30 HapMap Yoruban trios and 34 Estonian families. For 93.6% of the CNV loci, PiCNV enabled to unambiguously phase normal and CNV-carrying haplotypes and follow their transmission in the corresponding families. Furthermore, allelic composition analysis identified the co-occurrence of alternative allelic copies within 66.7% of haplotypes carrying copy number gains. We also observed less frequent transmission of CNV-carrying haplotypes from parents to children compared to normal haplotypes and identified an emergence of several de novo deletions and duplications in the offspring.

Detection of tmRNA molecules on microarrays at low temperatures using helper oligonucleotides

BackgroundThe hybridization of synthetic Streptococcus pneumoniae tmRNA on a detection microarray is slow at 34°C resulting in low signal intensities.ResultsWe demonstrate that adding specific DNA helper oligonucleotides (chaperones) to the hybridization buffer increases the signal strength at a given temperature and thus makes the specific detection of Streptococcus pneumoniae tmRNA more sensitive. No loss of specificity was observed at low temperatures compared to hybridization at 46°C. The effect of the chaperones can be explained by disruption of the strong secondary and tertiary structure of the target RNA by the selective hybridization of helper molecules. The amplification of the hybridization signal strength by chaperones is not necessarily local; we observed increased signal intensities in both local and distant regions of the target molecule.ConclusionsThe sensitivity of the detection of tmRNA at low temperature can be increased by chaperone oligonucleotides. Due to the complexity of RNA secondary and tertiary structures the effect of any individual chaperone is currently not predictable.