A bioinformatic filter for improved base-call accuracy and polymorphism detection using the Affymetrix GeneChip® whole-genome resequencing platform

DNA resequencing arrays enable rapid acquisition of high-quality sequence data. This technology represents a promising platform for rapid high-resolution genotyping of microorganisms. Traditional array-based resequencing methods have relied on the use of specific PCR-amplified fragments from the query samples as hybridization targets. While this specificity in the target DNA population reduces the potential for artifacts caused by cross-hybridization, the subsampling of the query genome limits the sequence coverage that can be obtained and therefore reduces the technique's resolution as a genotyping method. We have developed and validated an Affymetrix Inc. GeneChip® array-based, whole-genome resequencing platform for Francisella tularensis, the causative agent of tularemia. A set of bioinformatic filters that targeted systematic base-calling errors caused by cross-hybridization between the whole-genome sample and the array probes and by deletions in the sample DNA relative to the chip reference sequence were developed. Our approach eliminated 91% of the false-positive single-nucleotide polymorphism calls identified in the SCHU S4 query sample, at the cost of 10.7% of the true positives, yielding a total base-calling accuracy of 99.992%

Meraculous: De Novo Genome Assembly with Short Paired-End Reads

We describe a new algorithm, meraculous, for whole genome assembly of deep paired-end short reads, and apply it to the assembly of a dataset of paired 75-bp Illumina reads derived from the 15.4 megabase genome of the haploid yeast Pichia stipitis. More than 95% of the genome is recovered, with no errors; half the assembled sequence is in contigs longer than 101 kilobases and in scaffolds longer than 269 kilobases. Incorporating fosmid ends recovers entire chromosomes. Meraculous relies on an efficient and conservative traversal of the subgraph of the k-mer (deBruijn) graph of oligonucleotides with unique high quality extensions in the dataset, avoiding an explicit error correction step as used in other short-read assemblers. A novel memory-efficient hashing scheme is introduced. The resulting contigs are ordered and oriented using paired reads separated by ∼280 bp or ∼3.2 kbp, and many gaps between contigs can be closed using paired-end placements. Practical issues with the dataset are described, and prospects for assembling larger genomes are discussed

Improvements to the Illumina Sequencing System and New Applications

Next Generation sequencing products and procedures for the Illumina platform were evaluated, several of which immediately benefited JGI users. Paired end-reads of 35 bases each have become routine on our new GAii instruments with output in excess of 6 billion bases per run. Single reads have been used for polishing finished genomic sequence data for over 2 years, and collaborative work has progressed to include genomic SNP detection, expression analysis for eukaryotes and prokaryotes, and ChIP DNA sequencing. Paired End-Reads have been particularly useful for SNP detection. Our beta testing activities include positive results for barcode libraries (indexing) using Illumina's 3 primer scheme, large-gap libraries produced several ways, and accurate 75 base read-length. Key technologies include improvements to the instruments, reagents, software, and information management tools

Correlation of Blood and Salivary pH Levels in Healthy, Gingivitis, and Periodontitis Patients before and after Non-Surgical Periodontal Therapy

Periodontitis is an infectious illness which leads to the inflammation of protective tissues around the teeth and the continuous loss of alveolar bone and conjunctive tissue. Biomarker analysis in serum and saliva helps in the evaluation of disease progression and activity. It is also established that every inflammatory change along with resultant damage of tissues ends up in altered pH values in the fluids and tissues. Aim: To correlate the connection of pH levels in both blood as well as saliva in healthy, periodontitis, and gingivitis patients. Materials and Methods: The current research involved 145 subjects amidst the age of 20 and 55 years. The subjects were split into three different groups: healthy (Group A), gingivitis (Group B), and finally chronic periodontitis (Group C). The recording of clinical parameters was done by gingival index (GI), probing depth (PD), and plaque index (PI). pH of saliva and blood was analyzed with the help of digital single electrode pH meter. Subjects have gone through scaling and root planning (SRP) coupled with the instructions of oral hygiene. They were recalled post 4 weeks, and saliva and blood samples were gathered for analyzing pH. Results: Clinical parameters GI and PI were statistically important in both group C as well as group B post SRP. A crucial change has been observed in attachment levels (AL) and PD in the case of periodontitis group post SRP. The difference in the salivary pH values were significant between group B vs. C and A vs. C before the treatment because the values for group C were acidic, whereas in groups B and A the pH was alkaline. After the treatment, the values were still significant because the pH has become more alkaline compared to preoperative value in both group B and C. Saliva’s pH levels have demonstrated a statistically significant reduction in group C post SRP. Conclusion: Salivary pH levels and blood evidently became alkaline in the group C patients post SRP and there is a positive correlation between them and the clinical parameters