Phred Quality Research Articles

Benefits of merging paired-end reads before pre-processing environmental metagenomics data.

High throughput sequencing of environmental DNA has applications in biodiversity monitoring, taxa abundance estimation, understanding the dynamics of community ecology, and marine species studies and conservation. Environmental DNA, especially, marine eDNA, has a fast degradation rate. Aside from the good quality reads, the data could have a significant number of reads that fall slightly below the default PHRED quality threshold of 30 on sequencing. For quality control, trimming methods are employed, which generally precede the merging of the read pairs. However, in the case of eDNA, a significant percentage of reads within the acceptable quality score range are also dropped. To infer the ideal merge tool that is sensitive to eDNA, two Hiseq paired-end eDNA datasets were utilized to study the merging by the tools - FLASH (Fast Length Adjustment of SHort reads), PANDAseq, COPE, BBMerge, and VSEARCH without preprocessing. We assessed these tools on the following parameters: Time taken to process, the quality, and the number of merged reads. Trimmomatic, a widely-used preprocessing tool, was also assessed by preprocessing the datasets at different parameters for the two approaches of preprocessing: Sliding Window and Maximum Information. The preprocessed read pairs were then merged using the ideal merge tool identified earlier. FLASH is the most efficient merge tool balancing data conservation, quality of reads, and processing time. We compared Trimmomatic's two quality trimming options with increasing strictness with FLASH's direct merge. The raw reads processed with Trimmomatic then merged, yielded a significant drop in reads compared to the direct merge. An average of 29% of reads was dropped when directly merged with FLASH. Maximum Information option resulted in 30.7% to 68.05% read loss with lowest and highest stringency parameters, respectively. The Sliding Window approach conserves approximately 10% more reads at a PHRED score of 25 set as the threshold for a window of size 4. The lowered PHRED cut off conserves about 50% of the reads that could potentially be informative. We noted no significant reduction of data while optimizing the number of reads read in a window with the ideal quality (Q) score. Losing reads can negatively impact the downstream processing of the environmental data, especially for sequence alignment studies. The quality trim-first-merge-later approach can significantly decrease the number of reads conserved. However, direct merging of pair-end reads using FLASH conserved more than 60% of the reads. Therefore, direct merging of the paired-end reads can prevent potential removal of informative reads that do not comply by the trimming tool's strict checks. FLASH to be an efficient tool in conserving reads while carrying out quality trimming in moderation. Overall, our results show that merging paired-end reads of eDNA data before trimming can conserve more reads.

Germline and Somatic Variants Co-Ocurrence Profile in Early Onset Adult Myelodysplastic Syndromes without a Preexisting Disorder

Germline and Somatic Variants Co-Ocurrence Profile in Early Onset Adult Myelodysplastic Syndromes without a Preexisting Disorder

Nanopore Single-Molecule Sequencing for Mitochondrial DNA Methylation Analysis: Investigating Parkin-Associated Parkinsonism as a Proof of Concept.

Objective: To establish a workflow for mitochondrial DNA (mtDNA) CpG methylation using Nanopore whole-genome sequencing and perform first pilot experiments on affected Parkin biallelic mutation carriers (Parkin-PD) and healthy controls.Background: Mitochondria, including mtDNA, are established key players in Parkinson's disease (PD) pathogenesis. Mutations in Parkin, essential for degradation of damaged mitochondria, cause early-onset PD. However, mtDNA methylation and its implication in PD is understudied. Herein, we establish a workflow using Nanopore sequencing to directly detect mtDNA CpG methylation and compare mtDNA methylation between Parkin-related PD and healthy individuals.Methods: To obtain mtDNA, whole-genome Nanopore sequencing was performed on blood-derived from five Parkin-PD and three control subjects. In addition, induced pluripotent stem cell (iPSC)-derived midbrain neurons from four of these patients with PD and the three control subjects were investigated. The workflow was validated, using methylated and unmethylated 897 bp synthetic DNA samples at different dilution ratios (0, 50, 100% methylation) and mtDNA without methylation. MtDNA CpG methylation frequency (MF) was detected using Nanopolish and Megalodon.Results: Across all blood-derived samples, we obtained a mean coverage of 250.3X (SD ± 80.5X) and across all neuron-derived samples 830X (SD ± 465X) of the mitochondrial genome. We detected overall low-level CpG methylation from the blood-derived DNA (mean MF ± SD = 0.029 ± 0.041) and neuron-derived DNA (mean MF ± SD = 0.019 ± 0.035). Validation of the workflow, using synthetic DNA samples showed that highly methylated DNA molecules were prone to lower Guppy Phred quality scores and thereby more likely to fail Guppy base-calling. CpG methylation in blood- and neuron-derived DNA was significantly lower in Parkin-PD compared to controls (Mann-Whitney U-test p < 0.05).Conclusion: Nanopore sequencing is a useful method to investigate mtDNA methylation architecture, including Guppy-failed reads is of importance when investigating highly methylated sites. We present a mtDNA methylation workflow and suggest methylation variability across different tissues and between Parkin-PD patients and controls as an initial model to investigate.

Germinal Predisposition in Myelodysplastic Syndromes in Young Adults without a Preexisting Disorder or Organ Dysfunction: Identification of Deleterious Variants in Microsatellite Instability Genes

Germinal Predisposition in Myelodysplastic Syndromes in Young Adults without a Preexisting Disorder or Organ Dysfunction: Identification of Deleterious Variants in Microsatellite Instability Genes

Transcriptome sequencing assisted discovery and computational analysis of novel SNPs associated with flowering in Raphanus sativus in-bred lines for marker-assisted backcross breeding

The sequencing of radish genome aids in the better understanding and tailoring of traits associated with economic importance. In order to accelerate the genomics assisted breeding and genetic selection, transcriptomes of 33 radish inbred lines with diverse traits were sequenced for the development of single nucleotide polymorphic (SNP) markers. The sequence reads ranged from 2,560,543,741 bp to 20,039,688,139 bp with the GC (%) of 47.80–49.34 and phred quality score (Q30) of 96.47–97.54%. A total of 4951 polymorphic SNPs were identified among the accessions after stringent filtering and 298 SNPs with efficient marker assisted backcross breeding (MAB) markers were generated from the polymorphic SNPs. Further, functional annotations of SNPs revealed the effects and importance of the SNPs identified in the flowering process. The SNPs were predominantly associated with the four major flowering related transcription factors such as MYB, MADS box (AG), AP2/EREB, and bHLH. In addition, SNPs in the vital flowering integrator gene (FT) and floral repressors (EMBRYONIC FLOWER 1, 2, and FRIGIDA) were identified among the radish inbred lines. Further, 50 SNPs were randomly selected from 298 SNPs and validated using Kompetitive Allele Specific PCR genotyping system (KASP) in 102 radish inbred lines. The homozygosity of the inbred lines varied from 56 to 96% and the phylogenetic analysis resulted in the clustering of inbred lines into three subgroups. Taken together, the SNP markers identified in the present study can be utilized for the discrimination, seed purity test, and adjusting parental combinations for breeding in radish.

Adipose Tissue Gene Expression Associations Reveal Hundreds of Candidate Genes for Cardiometabolic Traits

Genome-wide association studies (GWASs) have identified thousands of genetic loci associated with cardiometabolic traits including type 2 diabetes (T2D), lipid levels, body fat distribution, and adiposity, although most causal genes remain unknown. We used subcutaneous adipose tissue RNA-seq data from 434 Finnish men from the METSIM study to identify 9,687 primary and 2,785 secondary cis-expression quantitative trait loci (eQTL; <1 Mb from TSS, FDR < 1%). Compared to primary eQTL signals, secondary eQTL signals were located further from transcription start sites, had smaller effect sizes, and were less enriched in adipose tissue regulatory elements compared to primary signals. Among 2,843 cardiometabolic GWAS signals, 262 colocalized by LD and conditional analysis with 318 transcripts as primary and conditionally distinct secondary cis-eQTLs, including some across ancestries. Of cardiometabolic traits examined for adipose tissue eQTL colocalizations, waist-hip ratio (WHR) and circulating lipid traits had the highest percentage of colocalized eQTLs (15% and 14%, respectively). Among alleles associated with increased cardiometabolic GWAS risk, approximately half (53%) were associated with decreased gene expression level. Mediation analyses of colocalized genes and cardiometabolic traits within the 434 individuals provided further evidence that gene expression influences variant-trait associations. These results identify hundreds of candidate genes that may act in adipose tissue to influence cardiometabolic traits.

Germline and Acquired Genetic Variants in Myelodysplastic Syndromes in Young Adults without a Preexisting Disorder or Organ Dysfunction

Background and Aim:It is increasingly recognized that patients with a de novomyelodysplastic syndrome (MDS) onset as young adults, lacking any other feature of a congenital disorder, may share a pathogenic overlap due to the presence of both germline and somatic variants. Identifying an inherited pathogenic variant has important therapeutic implications beyond family counselling: adapting the selection of sibling donor, the use of highly cytotoxic therapy and the monitoring for other cancer development. However, most studies have focused on patients with suspected inherited disorders based on the presence of physical abnormalities and/or family history. In addition, a mixture of pediatric and adult cases is usually reported. The aim of this study is to characterize the germline and tumor variants in a group of adult MDS patients without accompanying congenital physical anomalies and or family antecedent of bone marrow failure.Methods: We included 72 patients from 15 Spanish centers with a diagnosis of MDS between 18 and 60 years old (y.o). Patients with a previously diagnosed or suspected (one physical anomaly or family history) congenital syndrome were excluded. Diagnoses were made in accordance with the WHO 2016 classification. Whole-exome sequencing (WES) libraries were prepared using SureSelectXT Target Enrichment and sequenced on a HiSeq4000 platform (IlluminaInc.). Mean number of reads per sample was 138,726,017 with a Phred Quality Score >30 in 95.05% of bases. Read mapping sequence alignment and variant calling were performed using Biomedical Workbench (Qiagen). WES was performed on 72 tumor and 32 paired germinal DNA (buccal swab). To identify potential germline-causal mutations, a selection tool was implemented incorporating 239 genes associated with cause or predisposition to bone marrow failure or cancer. Variants with an ExAC, TOPMed and/or European 1000 Genomes minor allele frequency ≥0.01 were discarded.Results: The median age at diagnosis was 49 y.o. The cohort was categorised into two groups, less or equal 50 y.o. (62.5%) and between 50 and 60 y.o. (37.5%). In the first group, the frequency according to the WHO classification were 12% MDS with single lineage dyplasia (MDS-SLD), 8% MDS with ring sideroblasts (MDS-RS), 11% MDS with multilineage dyplasia (MDS-MLD), 24% MDS with excess blasts(MD-EB), 4% MDS with isolated del(5q)(MDS-del5q), 4% MDS unclassifiable and 4% chronic myelomonocytic leukemia (CMML). Meanwhile, in the group with age more than 50 y.o., the subtypes were 3.7% MDS-SLD, 7.4% MDS-RS, 29.6% MDS-MLD, 40.7% MD-EB, 3.7% MDS-del5q, and 14.8% CMML.Patients less or equal 50 y.o. were stratified based on IPSS-R as very low (4%), low (64%), intermediate (20%), high (12%) and very high (0%); and the group of more than 50 y.o. as very low (14.8%), low (33.3%), intermediate (29.6%), high (11.1%) and very high (11.1%).The mean number of somatic mutations was 0.68 in patients with less or equal 50 y.o. and 1.37 in those between 50 and 60 y.o., p=0.033 (U Mann-Whitney); and regarding germline variants, the first group mean number was 2.44 (p25-75, 1-3) and the second group showed a mean of 1.85 (QI 25-75, 1-3), p= 0,331.In the whole cohort, germline variants were found in 62 out of 72 patients, with the following frequencies: ATR(N=5, 6.9%), followed by BARD1(N=5, 6.9%), ERCC6L2(N=4, 5.6%), MSH6(N=4, 5.6%), TCIRG1(N=4, 5.6%), NBEAL2(N=4, 5.6%), ASXL1(N=3, 4.2%), ATM(N=3, 4.2%), MPL(N=3, 4.2%), NF1(N=3, 4.2%), RECQL4(N=3, 4.2%), SAMD9L(N=3, 4.2%), WRN(N=3, 4.2%).Among germline variants, those reported previously as pathogenic or likely pathogenic, or involving genes associated with familial MDS/AML included: ERCC6L2(N=4, 5.6%), SAMD9L(N=3, 4.2%), and one case mutated for DDX41, FANCC, JAK2, MSH6, SETBP1, MUTYH, BRCA1and RECQL4. In the whole cohort, somatic variants were found in 38 patients, with the following frequencies: TP53(N=7, 9.7%), ASXL1(N=7, 9.7%), SETBP1(N=5, 6.9%), NF1(N=5, 6.9%), SRSF2(N=4, 5.5%).Conclusion:In this subset of young adults with de novo MDS without congenital anomalies and/or familial history suggesting the presence of an undiagnosed congenital syndrome, 18% of the cohort harbored a likely causative germline variant. In addition, we noted a predominance of variants affecting genes with a cancer predisposition limited to the hematopoietic system, rather than classical telomere, DNA damage genes with an established mendelian link. [Display omitted] DisclosuresDíez-Campelo:Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau.

An improved filtering algorithm for big read datasets and its application to single-cell assembly

BackgroundFor single-cell or metagenomic sequencing projects, it is necessary to sequence with a very high mean coverage in order to make sure that all parts of the sample DNA get covered by the reads produced. This leads to huge datasets with lots of redundant data. A filtering of this data prior to assembly is advisable. Brown et al. (2012) presented the algorithm Diginorm for this purpose, which filters reads based on the abundance of their k-mers.MethodsWe present Bignorm, a faster and quality-conscious read filtering algorithm. An important new algorithmic feature is the use of phred quality scores together with a detailed analysis of the k-mer counts to decide which reads to keep.ResultsWe qualify and recommend parameters for our new read filtering algorithm. Guided by these parameters, we remove in terms of median 97.15% of the reads while keeping the mean phred score of the filtered dataset high. Using the SDAdes assembler, we produce assemblies of high quality from these filtered datasets in a fraction of the time needed for an assembly from the datasets filtered with Diginorm.ConclusionsWe conclude that read filtering is a practical and efficient method for reducing read data and for speeding up the assembly process. This applies not only for single cell assembly, as shown in this paper, but also to other projects with high mean coverage datasets like metagenomic sequencing projects.Our Bignorm algorithm allows assemblies of competitive quality in comparison to Diginorm, while being much faster. Bignorm is available for download at https://git.informatik.uni-kiel.de/axw/Bignorm.

Validating sonication as a DNA extraction method for use with carrion flies

Entomological evidence can be critical in establishing a postmortem interval estimate. DNA-based species identification can be an extremely valuable tool for forensic entomology. The problem of processing samples in a consistent, cost-effective manner that retains the morphological attributes of the specimen for vouchering has led us to investigate sonication as a primary means of non-destructive DNA extraction from carrion flies. We analyze the efficacy of this technique and compare it to an established DNA extraction technique — the Qiagen DNeasy tissue kit. Our results indicate that sonication produces a significant reduction in the sequence length and lower PHRED quality scores when compared to sequences using DNA obtained using the DNeasy kit, but species identification and phylogenetic inferences between sonication and DNeasy extractions are equivalent.

MEEPTOOLS: a maximum expected error based FASTQ read filtering and trimming toolkit

Quality-based sequence trimming of next generation sequencing (NGS) data is essential as it can potentially prevent missed or incorrect alignment downstream or prevent missassembly in a de novo assembly process by over-representing false k-mers. Trimming algorithms in tools like sickle, trimmomatic, etc. either depend on a running sum of quality of bases; or rely on average base quality of a sliding window. They consider PHRED quality, which is exponentially related to the probability of an erroneous base call. Here we present MEEPTOOLS, which is an open-source tool based on maximum expected error (MEE) as a percentage of read length (MEEP score) to filter, trim, truncate and assess NGS data in FASTQ file. By treating read trimming as a minimum subarray problem, MEEPTOOLS can simultaneously retain more reliable bases and remove unreliable bases than the traditional quality filtering strategies. MEEPTOOLS is ready available for download under the GNU GPLv3 at https://github.com/nisheth/meeptools.

Comparison of the performance of Ion Torrent chips in noninvasive prenatal trisomy detection.

Semiconductor high-throughput sequencing, represented by Ion Torrent PGM/Proton, proves to be feasible in the noninvasive prenatal diagnosis of fetal aneuploidies. It is commendable that, with less data and relevant cost also, an accurate result can be achieved owing to the high sensitivity and specificity of such kind of technology. We conducted a comparative analysis of the performance of four different Ion chips in detecting fetal chromosomal aneuploidies. Eight maternal plasma DNA samples, including four pregnancies with normal fetuses and four with trisomy 21 fetuses, were sequenced on Ion Torrent 314/316/318/PI chips, respectively. Results such as read mapped ratio, correlation coefficient and phred quality score were calculated and parallelly compared. All samples were correctly classified even with low-throughput chip, and, among the four chips, the 316 chip had the highest read mapped ratio, correlation coefficient, mean read length and phred quality score. All chips were well consistent with each other. Our results showed that all Ion chips are applicable in noninvasive prenatal fetal aneuploidy diagnosis. We recommend researchers or clinicians to use the appropriate chip with barcoding technology on the basis of the sample number.

AlienTrimmer removes adapter oligonucleotides with high sensitivity in short-insert paired-end reads. Commentary on Turner (2014) Assessment of insert sizes and adapter content in FASTQ data from NexteraXT libraries.

AlienTrimmer removes adapter oligonucleotides with high sensitivity in short-insert paired-end reads. Commentary on Turner (2014) Assessment of insert sizes and adapter content in FASTQ data from NexteraXT libraries.

Identification of Novel Point Mutations in Splicing Sites by the Integration of Exome and RNA Sequencing Data in Myeloproliferative Diseases

Abstract 2462Point mutations in intronic regions near mRNA splice junctions can affect mRNA splicing, altering the resulting RNA sequence.The molecular characterization of in-frame or out-of-frame splicing variants in cancer samples can potentially assist in the molecular characterization of tumors.The aim of this study was to identify mutations located in the 5' or 3' exon-intron borders that affect RNA splicing using whole-exome sequencing analysis, a technique that targets coding sequences but also include the nearby intronic regions.In order to identify novel (in-frame and out-of-frame) splicing variants in myeloproliferative disorders we developed a bioinformatics procedure ‘Splice-Site Prediction Procedure to analyze Next Generation Sequencing data' (SSPP-NGS).The SSPP-NGS bioinformatics method is an integration of two functional annotation tools for high-throughput sequencing data, ANNOVAR and MutationTaster and two canonical splice-site analysis tools, NetGene2 and Neural Network Promoter Prediction Tool (NNPPT).In addition, to assess the phenotypic effects of intronic mutations on mRNA splicing we combined DNA mutational screening analysis with RNA-Seq mediated gene expression profiling. Whole genome expression analysis was performed by using TopHat and Cufflinks: the first one is a splice junction mapper for RNA-Seq experiments able to mapp the reads against the junction to confirm them; the second one estimates gene expression, isoform-level expression, transcript abundance, differential gene expression and splicing.We used ANNOVAR and MutationTaster based on statistical Naive Bayes classifier to predict the non-coding mutations that affected physiological splicing. We then confirmed the results by queering NetGene2 and NNPPT using default parameters. Only the predictions found in all three programs were accepted as putative splicing variants and sequenced by Sanger method.We applied the entire procedure to whole exome sequencing data from 1 Ph+ leukemic patient sample (>80% myeloid cells) matched to autologous normal lymphocytes: on average, 70 million of paired-end reads and 5.2 gigabases (Gb) of sequences were generated per sample. A total of 177 candidate somatic point mutations (with minimum read depth of 20, minimum percent of substitution equal to 25% and minimum average Phred quality score of 30, corresponding to an accuracy of 99.9%, confirmed by at least 6 individual sequences) were found: 82/177 annotated in coding regions and 95/177 in non-coding regions. In particular 5/95 were located within 10-bp from a splicing junction. SSPS-NGS prediction analysis suggested the presence of 1/5 potential splicing site (predicting a loss of physiologic donor splicing site), while 4/5 were annotated as polymorphisms. The hypothetical splicing variant was located near the 5’ donor splice site at position +1 in the intron between exons 5 and 6 of the GNAQ gene (IVS5+1C->T); it was present with a frequency of mutation of 35%, corresponding to its heterozygous presence in 88% of cells. The presence of this heterozygous mutation was confirmed by Sanger method. SSPS-NGS allowed us to focus on transcriptional analysis of this gene. RNA-seq analysis showed that 73% of GNAQ mRNA effectively skipped the upstream exon 5, resulting in a 4 to 6 frameshift fusion, which likely destroys the GTPase activity of GNAQ. No evidence of GNAQ exon 5 deleted RNA was found in additional 7 patients analyzed who lacked the intronic mutation.We extended the SSPN-NGS analysis to 7 myeloproliferative patients analyzed by exome sequencing. Three novel heterozygous splicing variants were identified, affecting the HOOK1, SMAD9 and DNAH9 genes. All mutations were confirmed by Sanger method. SSPS-NSG analysis predicted 1 loss of donor site in-frame (DNAH9) and 2 loss of acceptor splice site out of frame (HOOK1 and SMAD9), in one case with an activation of a new cryptic splicing site (HOOK1). RNA-seq analysis is in progress.In conclusion, the work presented here showed the applicability of SSPPs-NGS to whole-exome sequencing data as a tool to complement exome analysis, in order to identify novel splicing variants. Disclosures:No relevant conflicts of interest to declare.

A protocol for a single-cell PCR of diatoms from fixed samples: method validation using Ditylum brightwellii (T. West) Grunow

Many major advances in macro-organismal biology, population genetics and evolutionary biology may be attributed to the development of rapid and inexpensive molecular methodology. Here, a simple protocol is presented for a polymerase chain reaction (PCR) of alcohol-preserved single diatom cells that facilitate studies requiring a large number of individuals, inclusive of archived and uncultivable specimens. Our approach integrates cell isolation and PCR amplification of two molecular markers, as well as post-PCR identification by scanning electron microscopy, thereby bridging the gap between classical and molecular diatom biology. The amplification of two genes was performed in a two-step protocol. By applying multiple sets of PCR primers to a single diatom cell, an internal fragment of the large subunit of the ribulose-1,5-bisphosphate carboxylase gene (RuBisCO, rbcL) was successfully amplified and sequenced, as well as the internal transcribed spacer (ITS) region in 20% of the same, preserved cells derived from natural phytoplankton. Multiple-gene recovery from a single-cell source requires further optimization, likely specific for the taxa of interest. Single marker recovery (from first-step amplification to sequencing) from natural, fixed single-cell samples was more successful with overall success rate of ∼31% for ITS and 67% for rbcL, respectively. Admittedly it is 50–70% lower than rates for a culture-pelleted DNA source (68% for ITS and 100% for rbcL) using current standard methods of sequence recovery in diatom studies, but our method is much less labor- and cost-intensive. This approach was applied to the diatom Ditylum brightwellii to assess the quality of recovered sequences. The phred quality scores of our sequences derived from single-cell material were similar to phred scores of our own, published sequences obtained from cultures grown in our laboratory.

ESTs from Seeds to Assist the Selective Breeding of Jatropha curcas L. for Oil and Active Compounds.

We report here on the characterization of a cDNA library from seeds of Jatropha curcas L. at three stages of fruit maturation before yellowing. We sequenced a total of 2200 clones and obtained a set of 931 non-redundant sequences (unigenes) after trimming and quality control, ie, 140 contigs and 791 singlets with PHRED quality ≥10. We found low levels of sequence redundancy and extensive metabolic coverage by homology comparison to GO. After comparison of 5841 non-redundant ESTs from a total of 13193 reads from GenBank with KEGG, we identified tags with nucleotide variations among J. curcas accessions for genes of fatty acid, terpene, alkaloid, quinone and hormone pathways of biosynthesis. More specifically, the expression level of four genes (palmitoyl-acyl carrier protein thioesterase, 3-ketoacyl-CoA thiolase B, lysophosphatidic acid acyltransferase and geranyl pyrophosphate synthase) measured by real-time PCR proved to be significantly different between leaves and fruits. Since the nucleotide polymorphism of these tags is associated to higher level of gene expression in fruits compared to leaves, we propose this approach to speed up the search for quantitative traits in selective breeding of J. curcas. We also discuss its potential utility for the selective breeding of economically important traits in J. curcas.

Chromatin Immunoprecipitation of GFP-Tagged PML-Rara Coupled to High-Throughput Next Generation Sequencing.

Abstract 1276Poster Board I-298The PML-RARA fusion protein produced by the t(15;17) translocation is found in acute promyelocytic leukemia (APL) and acts as an aberrant transcription factor with oncogenic properties. To define the high affinity DNA binding sites of PML-RARA, we developed a novel system based upon chromatin immunoprecipitation of eGFP tagged PML-RARA (PR), coupled to next generation sequencing. Chromatin isolated from flow sorted, GFP+ PR9 cells (isolated 24 hours after electroporation with eGFP-PR) was immunoprecipitated with a highly specific anti-GFP monoclonal antibody, and massively parallel sequencing was performed using single-end read libraries generated from both input DNA and immunoprecipitated DNA. The sequenced reads were mapped to the reference human genome using the Burrows-Wheeler Alignment tool (BWA). Using the SamTools package, alignment files were then filtered to retain only reads with phred quality scores greater than 30. Finally, Model Based Analysis of ChIP-Seq (MACS) was used to obtain the predicted binding sites (approximately 13,000 for each replicate using a p value cutoff of 0.00001). To ensure the reproducibility of called peaks, we limited our analysis to 701 sites that occurred in replicate sequencing runs of the libraries, within a tolerance of ±50 bp at the peak. Visual inspection of graphically plotted peaks further filtered this list to 421 high quality sites. Using microarray expression data for 14 APL patients and 5 flow sorted, normal promyelocyte samples, we selected neighboring genes with at least a 3-fold difference between APL and promyelocyte expression. This step yielded a list of 82 neighboring genes whose expression may be altered by PML-RARA binding to adjacent DNA. While PML-RARA is often considered a transcriptional repressor, 68% of these genes were overexpressed in APL, suggesting that PML-RARA may also function as a transcriptional activator. Furthermore, 51 genes were dysregulated in the mCG-PML-RARA murine model of APL, 16 genes demonstrated expression changes following induction of PML-RARA expression in PR9 cells, and 12 genes had altered expression in both the PR9 and murine models. Collectively, these results demonstrate an association between the high confidence putative PR binding sites and gene expression changes. We next used the sequences found within the best 89 binding sites to define 6 potential in vivo consensus sites of PML-RARA using the CONSENSUS program. Three of these predicted sites were present in greater than 40% of the 421 high confidence sites. Two of these common motifs resemble the motifs found in retinoic acid response elements (RAREs), but have less stringent conservation at the 5' end while retaining the 3' TCA sequence. Taken together, our results suggest that PML-RARA has an extended repertoire of genomic DNA binding sites compared to wild-type RARA, reflecting novel gain-of-function properties of the fusion protein. Binding of some of these sites appears to have direct consequences for the expression of several tightly linked genes, which may themselves be involved in transcriptional regulatory networks that contribute to APL pathogenesis. DisclosuresNo relevant conflicts of interest to declare.

GRAT—genome-scale rapid alignment tool

Modern alignment methods designed to work rapidly and efficiently with large datasets often do so at the cost of method sensitivity. To overcome this, we have developed a novel alignment program, GRAT, built to accurately align short, highly similar DNA sequences. The program runs rapidly and requires no more memory and CPU power than a desktop computer. In addition, specificity is ensured by statistically separating the true alignments from spurious matches using phred quality values. An efficient separation is especially important when searching large datasets and whenever there are repeats present in the dataset. Results are superior in comparison to widely used existing software, and analysis of two large genomic datasets show the usefulness and scalability of the algorithm.

Benchmark for Evaluating the Quality of DNA Sequencing: Proposal from an International External Quality Assessment Scheme

In the past 15 years, clinical laboratory science has been transformed by the use of technologies that cross the traditional boundaries between laboratory disciplines. However, during this period, issues of quality have not always been given adequate attention. The European Molecular Genetics Quality Network (EMQN) has developed a novel external quality assessment scheme for evaluation of DNA sequencing. We report the results of an international survey of the quality of DNA sequencing among 64 laboratories from 21 countries. Current practice for DNA sequence analysis was established by use of an online questionnaire. Participating laboratories were provided with 4 DNA samples of validated genotype. Evaluation of the results included assessing the quality of sequence data, variant genotypes, and mutation nomenclature. To accommodate variations in mutation nomenclature, variants indicated by participants were scored for compliance with 3 acceptable marking schemes. A total of 346 genotypes were analyzed. Of these, 19 (5%) genotyping errors were made. Of these, 10 (53%) were false-negative and 9 (47%) were false-positive results. A further 27 (8%) errors were made in naming mutations. Results were analyzed for 3 indicators of data quality: PHRED quality scores, Quality Read Length, and Quality Read Overlap. Most laboratories produced results of acceptable diagnostic quality as judged by these indicators. The results were used to calculate a consensus benchmark for DNA sequencing against which individual laboratories could rank their performance. We propose that the consensus benchmark can be used as a baseline against which the aggregate and individual laboratory standard of DNA sequencing may be tracked from year to year.

Transcriptional Profiles of the Human Pathogenic Fungus Paracoccidioides brasiliensis in Mycelium and Yeast Cells

Paracoccidioides brasiliensis is the causative agent of paracoccidioidomycosis, a disease that affects 10 million individuals in Latin America. This report depicts the results of the analysis of 6,022 assembled groups from mycelium and yeast phase expressed sequence tags, covering about 80% of the estimated genome of this dimorphic, thermo-regulated fungus. The data provide a comprehensive view of the fungal metabolism, including overexpressed transcripts, stage-specific genes, and also those that are up- or down-regulated as assessed by in silico electronic subtraction and cDNA microarrays. Also, a significant differential expression pattern in mycelium and yeast cells was detected, which was confirmed by Northern blot analysis, providing insights into differential metabolic adaptations. The overall transcriptome analysis provided information about sequences related to the cell cycle, stress response, drug resistance, and signal transduction pathways of the pathogen. Novel P. brasiliensis genes have been identified, probably corresponding to proteins that should be addressed as virulence factor candidates and potential new drug targets.

Microchip bioprocessor for integrated nanovolume sample purification and DNA sequencing.

A microfabricated electrophoretic bioprocessor for integrated DNA sequencing sample desalting, template removal, preconcentration, and CE analysis is presented. A low-viscosity gel capture matrix, containing an acrylamide-copolymerized oligonucleotide complementary to the 20-base sequence directly 3' of the M13-40 universal forward priming site, is introduced into the 60-nL capture chamber. Unpurified DNA sequencing reaction products are electrophoretically driven through the chamber; extension products hybridize to the matrix, while contaminating buffering ions, Cl-, excess primer, and template DNA are unretained. Purification under optimized conditions is complete in only 120 s (binding temperature 50 degrees C, driving voltage 250 V). High-speed, integrated sequencing analysis is accomplished by releasing the gel-purified duplex at 67 degrees C and directly injecting onto a 15.9-cm effective length CE microchannel. Electrophoretic resolution of the sequencing products is complete in 32 min, producing a total of 560 bp with phred quality q > or = 20 (accuracy > or = 99%). This fully integrated nanoliter process decreases the purification time approximately 10-fold and the process volume approximately 100-fold while providing state-of-the-art sequencing results.