PathogenMIPer: a tool for the design of molecular inversion probes to detect multiple pathogens

BACKGROUND: Here we describe PathogenMIPer, a software program for designing molecular inversion probe (MIP) oligonucleotides for use in pathogen identification and detection. The software designs unique and specific oligonucleotide probes targeting microbial or other genomes. The tool tailors all probe sequence components (including target-specific sequences, barcode sequences, universal primers and restriction sites) and combines these components into ready-to-order probes for use in a MIP assay. The system can harness the genetic variability available in an entire genome in designing specific probes for the detection of multiple co-infections in a single tube using a MIP assay. RESULTS: PathogenMIPer can accept sequence data in FASTA file format, and other parameter inputs from the user through a graphical user interface. It can design MIPs not only for pathogens, but for any genome for use in parallel genomic analyses. The software was validated experimentally by applying it to the detection of human papilloma virus (HPV) as a model system, which is associated with various human malignancies including cervical and skin cancers. Initial tests of laboratory samples using the MIPs developed by the PathogenMIPer to recognize 24 different types of HPVs gave very promising results, detecting even a small viral load of single as well as multiple infections (Akhras et al, personal communication). CONCLUSION: PathogenMIPer is a software for designing molecular inversion probes for detection of multiple target DNAs in a sample using MIP assays. It enables broader use of MIP technology in the detection through genotyping of pathogens that are complex, difficult-to-amplify, or present in multiple subtypes in a sample

Identification of rare DNA variants in mitochondrial disorders with improved array-based sequencing.

A common goal in the discovery of rare functional DNA variants via medical resequencing is to incur a relatively lower proportion of false positive base-calls. We developed a novel statistical method for resequencing arrays (SRMA, sequence robust multi-array analysis) to increase the accuracy of detecting rare variants and reduce the costs in subsequent sequence verifications required in medical applications. SRMA includes single and multi-array analysis and accounts for technical variables as well as the possibility of both low- and high-frequency genomic variation. The confidence of each base-call was ranked using two quality measures. In comparison to Sanger capillary sequencing, we achieved a false discovery rate of 2% (false positive rate 1.2 × 10⁻⁵, false negative rate 5%), which is similar to automated second-generation sequencing technologies. Applied to the analysis of 39 nuclear candidate genes in disorders of mitochondrial DNA (mtDNA) maintenance, we confirmed mutations in the DNA polymerase gamma POLG in positive control cases, and identified novel rare variants in previously undiagnosed cases in the mitochondrial topoisomerase TOP1MT, the mismatch repair enzyme MUTYH, and the apurinic-apyrimidinic endonuclease APEX2. Some patients carried rare heterozygous variants in several functionally interacting genes, which could indicate synergistic genetic effects in these clinically similar disorders

PathogenMip Assay: A Multiplex Pathogen Detection Assay

The Molecular Inversion Probe (MIP) assay has been previously applied to a large-scale human SNP detection. Here we describe the PathogenMip Assay, a complete protocol for probe production and applied approaches to pathogen detection. We have demonstrated the utility of this assay with an initial set of 24 probes targeting the most clinically relevant HPV genotypes associated with cervical cancer progression. Probe construction was based on a novel, cost-effective, ligase-based protocol. The assay was validated by performing pyrosequencing and Microarray chip detection in parallel experiments. HPV plasmids were used to validate sensitivity and selectivity of the assay. In addition, 20 genomic DNA extracts from primary tumors were genotyped with the PathogenMip Assay results and were in 100% agreement with conventional sequencing using an L1-based HPV genotyping protocol. The PathogenMip Assay is a widely accessible protocol for producing and using highly discriminating probes, with experimentally validated results in pathogen genotyping, which could potentially be applied to the detection and characterization of any microbe

Connector Inversion Probe Technology: A Powerful One-Primer Multiplex DNA Amplification System for Numerous Scientific Applications

We combined components of a previous assay referred to as Molecular Inversion Probe (MIP) with a complete gap filling strategy, creating a versatile powerful one-primer multiplex amplification system. As a proof-of-concept, this novel method, which employs a Connector Inversion Probe (CIPer), was tested as a genetic tool for pathogen diagnosis, typing, and antibiotic resistance screening with two distinct systems: i) a conserved sequence primer system for genotyping Human Papillomavirus (HPV), a cancer-associated viral agent and ii) screening for antibiotic resistance mutations in the bacterial pathogen Neisseria gonorrhoeae. We also discuss future applications and advances of the CIPer technology such as integration with digital amplification and next-generation sequencing methods. Furthermore, we introduce the concept of two-dimension informational barcodes, i.e. “multiplex multiplexing padlocks” (MMPs). For the readers' convenience, we also provide an on-line tutorial with user-interface software application CIP creator 1.0.1, for custom probe generation from virtually any new or established primer-pairs

Figure 6

<p>Schematic overview of the PathogenMip Assay. <b>A</b>) The 24 probes included in the assay are situated at their respective target sites on the approximately 8000 base pairs of double stranded HPV genomic DNA. Early genes (denoted E) code for virus integration and replication and late genes (denoted L) encode the viral capsule creation. The probes recognize ∼40 base pair fragments unique for each targeted genotype. <b>B</b>) Following enzymatic inversion of reacted probes and universal amplification, the amplicons are used for subsequent appropriate HPV genotype screening. <b>C</b>) Conventional HPV genotyping takes a different approach, in which the nested primer pairs PGMY09/11 and GP5+/6+ amplify respectively ∼450 base pair and ∼150 base pair fragments that, through an appropriate readout process, will make up the basis for genotyping. These primers are restricted to the highly conserved genomic regions, most commonly found in the L1 gene. <b>D</b>) Multiple-primer DNA Pyrosequencing of an incorporated ID-tag. The diagrams depict the complementary sequence of the investigated probes -16 and -18. Marked in the figure is the ID-tag for each probe and the point of ligation where the probes circularized, incorporation of a dGTP, seen here as the complementary “C”. <b>E</b>) The in-house barcode chips here used to detect one HPV-16 positive, and one HPV-18 positive in human genomic DNA presence as seen with a positive rMIP in both chips.</p

Figure 1

<p>Schematic overviews of molecular inversion probe technology. <b>A</b>) Synthetic oligonucleotide containing following four regions; i) H1 and H2: homology regions comprised of unique continuous 40–50 base pair fragments for target recognition ii) BARCODE: molecular barcode comprised of a 20 base pair DNA tag for target identification iii) U1 and U2: universal primer regions for inverted probe amplification, and iv) R: restriction site for probe linearization. <b>B</b>) Upon target recognition, a DNA polymerase fills the missing gap in between the juxtaposition of the probes' flanking ends, and through the activity of a DNA ligase the probe is circularized. In all cases the missing nucleotide is a “G”. <b>C</b>) Circular DNA enrichment through DNA degradation by enzymes Exonuclease I and III. <b>D</b>) Probe linearization restriction site cleavage. <b>E</b>) All reacted and inverted probes are amplified with universal primers, of which one is biotinylated for subsequent amplicon validation.</p

Figure 4

<p>Bar-histograms representing fluorescence intensities from the in-house barcode-chip for genotyping genomic DNA extracts from tumor samples derived from four patients with cervical cancers. Seen in the figure are four examples of single HPV infections, one from each genotype, observed in the sample set. HPV-16 was genotyped in sample OM-1751, HPV-18 in OM-1452, HPV-45 in OM-2258 and HPV-59 in OM-1569. The signal-intensities were normalized to the intensity of the peak for the reference probe targeting human β-globin gene (rMIP). The remaining bars constitute of the reaction background signal.</p

Figure 3

<p>Probe selectivity. The figure depicts superimposed pyrosequencing (dispensation time vs. light signal intensity) diagrams for two probes following the MIP reactions, targeting HPV-45 (black) and HPV-59 (red), mimicking a double HPV infection. <b>A</b>) The initial DNA amount of each contributing plasmid was 100 ng and equal levels of sequencing intensities are seen. <b>B</b>) The DNA amount of HPV-45 plasmid remained at 100 ng, while HPV-59 plasmid was set at 10 ng resulting in ∼2-fold lower signal intensity. <b>C</b>) DNA amounts were set at 100 ng for HPV-45 plasmid and 1 nanogram for HPV-59 plasmid, which was observed as a ∼3-fold decrease in signal intensity. <b>D</b>) DNA amounts of 100 ng of HPV-45 plasmid and 100 pg of HPV-59 plasmid resulted in signal from HPV-45-probe without a measurable signal from the probe for HPV-59.</p

Figure 2

<p>Ligase-based probe construction for the PathogenMip Assay. Seen in the figure is the sequence for probe-16 prior to inversion. Two shorter fragments were synthesized and hybridized to a bridge complementary to the universal primer regions, common for all probes used in the assay. The coupling of the shorter fragments was mediated by polymerase and ligase activity to achieve maximum yield.</p