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1.  Whole genome de novo assemblies of three divergent strains of rice, Oryza sativa, document novel gene space of aus and indica 
Genome Biology  2014;15(11):506.
Background
The use of high throughput genome-sequencing technologies has uncovered a large extent of structural variation in eukaryotic genomes that makes important contributions to genomic diversity and phenotypic variation. When the genomes of different strains of a given organism are compared, whole genome resequencing data are typically aligned to an established reference sequence. However, when the reference differs in significant structural ways from the individuals under study, the analysis is often incomplete or inaccurate.
Results
Here, we use rice as a model to demonstrate how improvements in sequencing and assembly technology allow rapid and inexpensive de novo assembly of next generation sequence data into high-quality assemblies that can be directly compared using whole genome alignment to provide an unbiased assessment. Using this approach, we are able to accurately assess the ‘pan-genome’ of three divergent rice varieties and document several megabases of each genome absent in the other two.
Conclusions
Many of the genome-specific loci are annotated to contain genes, reflecting the potential for new biological properties that would be missed by standard reference-mapping approaches. We further provide a detailed analysis of several loci associated with agriculturally important traits, including the S5 hybrid sterility locus, the Sub1 submergence tolerance locus, the LRK gene cluster associated with improved yield, and the Pup1 cluster associated with phosphorus deficiency, illustrating the utility of our approach for biological discovery. All of the data and software are openly available to support further breeding and functional studies of rice and other species.
Electronic supplementary material
The online version of this article (doi:10.1186/s13059-014-0506-z) contains supplementary material, which is available to authorized users.
doi:10.1186/s13059-014-0506-z
PMCID: PMC4268812  PMID: 25468217
2.  De novo Mutations in Schizophrenia Implicate Chromatin Remodeling and Support a Genetic Overlap with Autism and Intellectual Disability 
Molecular psychiatry  2014;19(6):652-658.
Schizophrenia is a serious psychiatric disorder with a broadly undiscovered genetic etiology. Recent studies of de novo mutations (DNM) in schizophrenia and autism have reinforced the hypothesis that rare genetic variation contributes to risk. We carried out exome sequencing on 57 trios with sporadic or familial schizophrenia. In sporadic trios, we observed a ~3.5-fold increase in the proportion of nonsense de novo mutations (DNMs) (0.101 vs. 0.031, empirical P=0.01, BH-corrected P=0.044). These mutations were significantly more likely to occur in genes with highly ranked probabilities of haploinsufficiency (P=0.0029, corrected P=0.006). DNMs of potential functional consequence were also found to occur in genes predicted to be less tolerant to rare variation (P=2.01×10−5, corrected P =2.1×10−3). Genes with DNMs overlapped with genes implicated in autism (e.g. AUTS2, CDH8, MECP2) and intellectual disability (ID) (e.g. HUWE1 and TRAPPC9), supporting a shared genetic etiology between these disorders. Functionally CHD8, MECP2 and HUWE1 converge on epigenetic regulation of transcription suggesting that this may be an important risk mechanism. Our results were consistent in an analysis of additional exome based sequencing studies of other neurodevelopmental disorders. These findings suggest that perturbations in genes which function in the epigenetic regulation of brain development and cognition could have a central role in the susceptibility to, pathogenesis, and treatment of mental disorders.
doi:10.1038/mp.2014.29
PMCID: PMC4031262  PMID: 24776741
De novo Mutations; Schizophrenia; Autism; CHD8; MECP2
3.  708 common and 2,010 rare DISC1 locus variants identified in 1,542 subjects: analysis for association with psychiatric disorder and cognitive traits 
Molecular psychiatry  2013;19(6):668-675.
A balanced t(1;11) translocation which transects the Disrupted in schizophrenia 1 (DISC1) gene shows genome-wide significant linkage for schizophrenia and recurrent major depressive disorder in a single large Scottish family, but genome-wide and exome sequencing-based association studies have not supported a role for DISC1 in psychiatric illness. To explore DISC1 in more detail, we sequenced 528 kb of the DISC1 locus in 653 cases and 889 controls. We report 2,718 validated single nucleotide polymorphisms of which 2,010 have a minor allele frequency of less than 1%. Only 38% of these variants are reported in the 1000 Genomes Project European subset. This suggests that many DISC1 SNPs remain undiscovered and are essentially private. Rare coding variants identified exclusively in patients were found in likely functional protein domains. Significant region-wide association was observed between rs16856199 and recurrent major depressive disorder (P=0.026, unadjusted P=6.3 × 10−5, OR=3.48). This was not replicated in additional recurrent major depression samples (replication P=0.11). Combined analysis of both the original and replication set supported the original association (P=0.0058, OR=1.46). Evidence for segregation of this variant with disease in families was limited to those of rMDD individuals referred from primary care. Burden analysis for coding and non-coding variants gave nominal associations with diagnosis and measures of mood and cognition. Together, these observations are likely to generalise to other candidate genes for major mental illness and may thus provide guidelines for the design of future studies.
doi:10.1038/mp.2013.68
PMCID: PMC4031635  PMID: 23732877
4.  Validation and assessment of variant calling pipelines for next-generation sequencing 
Human Genomics  2014;8(1):14.
Background
The processing and analysis of the large scale data generated by next-generation sequencing (NGS) experiments is challenging and is a burgeoning area of new methods development. Several new bioinformatics tools have been developed for calling sequence variants from NGS data. Here, we validate the variant calling of these tools and compare their relative accuracy to determine which data processing pipeline is optimal.
Results
We developed a unified pipeline for processing NGS data that encompasses four modules: mapping, filtering, realignment and recalibration, and variant calling. We processed 130 subjects from an ongoing whole exome sequencing study through this pipeline. To evaluate the accuracy of each module, we conducted a series of comparisons between the single nucleotide variant (SNV) calls from the NGS data and either gold-standard Sanger sequencing on a total of 700 variants or array genotyping data on a total of 9,935 single-nucleotide polymorphisms. A head to head comparison showed that Genome Analysis Toolkit (GATK) provided more accurate calls than SAMtools (positive predictive value of 92.55% vs. 80.35%, respectively). Realignment of mapped reads and recalibration of base quality scores before SNV calling proved to be crucial to accurate variant calling. GATK HaplotypeCaller algorithm for variant calling outperformed the UnifiedGenotype algorithm. We also showed a relationship between mapping quality, read depth and allele balance, and SNV call accuracy. However, if best practices are used in data processing, then additional filtering based on these metrics provides little gains and accuracies of >99% are achievable.
Conclusions
Our findings will help to determine the best approach for processing NGS data to confidently call variants for downstream analyses. To enable others to implement and replicate our results, all of our codes are freely available at http://metamoodics.org/wes.
doi:10.1186/1479-7364-8-14
PMCID: PMC4129436  PMID: 25078893
Variant calling pipelines; Next-generation sequencing; Exome sequencing
5.  Integrated RNA-seq and sRNA-seq analysis identifies novel nitrate-responsive genes in Arabidopsis thaliana roots 
BMC Genomics  2013;14:701.
Background
Nitrate and other nitrogen metabolites can act as signals that regulate global gene expression in plants. Adaptive changes in plant morphology and physiology triggered by changes in nitrate availability are partly explained by these changes in gene expression. Despite several genome-wide efforts to identify nitrate-regulated genes, no comprehensive study of the Arabidopsis root transcriptome under contrasting nitrate conditions has been carried out.
Results
In this work, we employed the Illumina high throughput sequencing technology to perform an integrated analysis of the poly-A + enriched and the small RNA fractions of the Arabidopsis thaliana root transcriptome in response to nitrate treatments. Our sequencing strategy identified new nitrate-regulated genes including 40 genes not represented in the ATH1 Affymetrix GeneChip, a novel nitrate-responsive antisense transcript and a new nitrate responsive miRNA/TARGET module consisting of a novel microRNA, miR5640 and its target, AtPPC3.
Conclusions
Sequencing of small RNAs and mRNAs uncovered new genes, and enabled us to develop new hypotheses for nitrate regulation and coordination of carbon and nitrogen metabolism.
doi:10.1186/1471-2164-14-701
PMCID: PMC3906980  PMID: 24119003
Arabidopsis; Nitrate; RNA-seq; Roots; MicroRNA; Transcriptomics
6.  Hybrid error correction and de novo assembly of single-molecule sequencing reads 
Nature biotechnology  2012;30(7):693-700.
Emerging single-molecule sequencing instruments can generate multi-kilobase sequences with the potential to dramatically improve genome and transcriptome assembly. However, the high error rate of single-molecule reads is challenging, and has limited their use to resequencing bacteria. To address this limitation, we introduce a novel correction algorithm and assembly strategy that utilizes shorter, high-identity sequences to correct the error in single-molecule sequences. We demonstrate the utility of this approach on Pacbio RS reads of phage, prokaryotic, and eukaryotic whole genomes, including the novel genome of the parrot Melopsittacus undulatus, as well as for RNA-seq reads of the corn (Zea mays) transcriptome. Our approach achieves over 99.9% read correction accuracy and produces substantially better assemblies than current sequencing strategies: in the best example, quintupling the median contig size relative to high-coverage, second-generation assemblies. Greater gains are predicted if read lengths continue to increase, including the prospect of single-contig bacterial chromosome assembly.
doi:10.1038/nbt.2280
PMCID: PMC3707490  PMID: 22750884
7.  Parallel comparison of Illumina RNA-Seq and Affymetrix microarray platforms on transcriptomic profiles generated from 5-aza-deoxy-cytidine treated HT-29 colon cancer cells and simulated datasets 
BMC Bioinformatics  2013;14(Suppl 9):S1.
Background
High throughput parallel sequencing, RNA-Seq, has recently emerged as an appealing alternative to microarray in identifying differentially expressed genes (DEG) between biological groups. However, there still exists considerable discrepancy on gene expression measurements and DEG results between the two platforms. The objective of this study was to compare parallel paired-end RNA-Seq and microarray data generated on 5-azadeoxy-cytidine (5-Aza) treated HT-29 colon cancer cells with an additional simulation study.
Methods
We first performed general correlation analysis comparing gene expression profiles on both platforms. An Errors-In-Variables (EIV) regression model was subsequently applied to assess proportional and fixed biases between the two technologies. Then several existing algorithms, designed for DEG identification in RNA-Seq and microarray data, were applied to compare the cross-platform overlaps with respect to DEG lists, which were further validated using qRT-PCR assays on selected genes. Functional analyses were subsequently conducted using Ingenuity Pathway Analysis (IPA).
Results
Pearson and Spearman correlation coefficients between the RNA-Seq and microarray data each exceeded 0.80, with 66%~68% overlap of genes on both platforms. The EIV regression model indicated the existence of both fixed and proportional biases between the two platforms. The DESeq and baySeq algorithms (RNA-Seq) and the SAM and eBayes algorithms (microarray) achieved the highest cross-platform overlap rate in DEG results from both experimental and simulated datasets. DESeq method exhibited a better control on the false discovery rate than baySeq on the simulated dataset although it performed slightly inferior to baySeq in the sensitivity test. RNA-Seq and qRT-PCR, but not microarray data, confirmed the expected reversal of SPARC gene suppression after treating HT-29 cells with 5-Aza. Thirty-three IPA canonical pathways were identified by both microarray and RNA-Seq data, 152 pathways by RNA-Seq data only, and none by microarray data only.
Conclusions
These results suggest that RNA-Seq has advantages over microarray in identification of DEGs with the most consistent results generated from DESeq and SAM methods. The EIV regression model reveals both fixed and proportional biases between RNA-Seq and microarray. This may explain in part the lower cross-platform overlap in DEG lists compared to those in detectable genes.
doi:10.1186/1471-2105-14-S9-S1
PMCID: PMC3697991  PMID: 23902433
8.  Current challenges in de novo plant genome sequencing and assembly 
Genome Biology  2012;13(4):243.
Genome sequencing is now affordable, but assembling plant genomes de novo remains challenging. We assess the state of the art of assembly and review the best practices for the community.
doi:10.1186/gb-2012-13-4-243
PMCID: PMC3446297  PMID: 22546054
DNA sequencing; genome assembly; plant genomics
9.  De Novo Gene Disruptions in Children on the Autistic Spectrum 
Neuron  2012;74(2):285-299.
SUMMARY
Exome sequencing of 343 families, each with a single child on the autism spectrum and at least one unaffected sibling, reveal de novo small indels and point substitutions, which come mostly from the paternal line in an age-dependent manner. We do not see significantly greater numbers of de novo missense mutations in affected versus unaffected children, but gene-disrupting mutations (nonsense, splice site, and frame shifts) are twice as frequent, 59 to 28. Based on this differential and the number of recurrent and total targets of gene disruption found in our and similar studies, we estimate between 350 and 400 autism susceptibility genes. Many of the disrupted genes in these studies are associated with the fragile X protein, FMRP, reinforcing links between autism and synaptic plasticity. We find FMRP-associated genes are under greater purifying selection than the remainder of genes and suggest they are especially dosage-sensitive targets of cognitive disorders.
doi:10.1016/j.neuron.2012.04.009
PMCID: PMC3619976  PMID: 22542183
10.  Chd5 Requires PHD-mediated Histone 3 Binding for Tumor Suppression 
Cell reports  2013;3(1):92-102.
SUMMARY
ChromodomainHelicase DNA-binding protein 5 (CHD5) is a tumor suppressor mapping to 1p36—a genomic region frequently deleted in human cancer. Although CHD5 belongs to the CHD family of chromatin remodeling proteins, whether its tumor suppressive role involves an interaction with chromatin is unknown. Here we report that Chd5 binds the unmodified N-terminus of H3 through its tandem plant homeodomains (PHDs). Genome-wide ChIP studies reveal preferential binding of Chd5 to loci lacking the active mark H3K4me3, and also identify novel Chd5-targets implicated in cancer. Chd5 mutations abrogating H3 binding are unable to inhibit proliferation or to transcriptionally modulate target genes, leading to tumorigenesis in vivo. Unlike wild-type Chd5, Chd5-PHD mutants are unable to induce differentiation or to efficiently suppress growth of human neuroblastoma in vivo. Our work defines Chd5 as an N-terminally unmodified H3-binding protein and provides functional evidence that this interaction orchestrates chromatin-mediated transcriptional programs critical for tumor suppression.
doi:10.1016/j.celrep.2012.12.009
PMCID: PMC3575599  PMID: 23318260
11.  A Hybrid Likelihood Model for Sequence-Based Disease Association Studies 
PLoS Genetics  2013;9(1):e1003224.
In the past few years, case-control studies of common diseases have shifted their focus from single genes to whole exomes. New sequencing technologies now routinely detect hundreds of thousands of sequence variants in a single study, many of which are rare or even novel. The limitation of classical single-marker association analysis for rare variants has been a challenge in such studies. A new generation of statistical methods for case-control association studies has been developed to meet this challenge. A common approach to association analysis of rare variants is the burden-style collapsing methods to combine rare variant data within individuals across or within genes. Here, we propose a new hybrid likelihood model that combines a burden test with a test of the position distribution of variants. In extensive simulations and on empirical data from the Dallas Heart Study, the new model demonstrates consistently good power, in particular when applied to a gene set (e.g., multiple candidate genes with shared biological function or pathway), when rare variants cluster in key functional regions of a gene, and when protective variants are present. When applied to data from an ongoing sequencing study of bipolar disorder (191 cases, 107 controls), the model identifies seven gene sets with nominal p-values0.05, of which one MAPK signaling pathway (KEGG) reaches trend-level significance after correcting for multiple testing.
Author Summary
Inexpensive, high-throughput sequencing has transformed the field of case-control association studies. For the first time, it may be possible to identify the genetic underpinnings of complex diseases, by sequencing the DNA of hundreds (even thousands) of cases and controls and comparing patterns of DNA sequence variation. However, complex diseases are likely to be caused by many variants, some of which are very rare. Taken one at a time, the association between variant and disease phenotype may not be detectable by current statistical methods. One strategy is to identify regions where important variants occur by “collapsing” variants into groups. Here, we present a new collapsing approach, capable of detecting subtle genetic differences between cases and controls. We show, in extensive simulations and using a benchmark set of genes involved in human triglyceride levels, that the approach is potentially more powerful than existing methods. We apply the new method to an ongoing sequencing study of bipolar cases and controls and identify a set of genes found in neuronal synapses, which may be implicated in bipolar disorder.
doi:10.1371/journal.pgen.1003224
PMCID: PMC3554549  PMID: 23358228
12.  SpliceTrap: a method to quantify alternative splicing under single cellular conditions 
Bioinformatics  2011;27(21):3010-3016.
Motivation: Alternative splicing (AS) is a pre-mRNA maturation process leading to the expression of multiple mRNA variants from the same primary transcript. More than 90% of human genes are expressed via AS. Therefore, quantifying the inclusion level of every exon is crucial for generating accurate transcriptomic maps and studying the regulation of AS.
Results: Here we introduce SpliceTrap, a method to quantify exon inclusion levels using paired-end RNA-seq data. Unlike other tools, which focus on full-length transcript isoforms, SpliceTrap approaches the expression-level estimation of each exon as an independent Bayesian inference problem. In addition, SpliceTrap can identify major classes of alternative splicing events under a single cellular condition, without requiring a background set of reads to estimate relative splicing changes. We tested SpliceTrap both by simulation and real data analysis, and compared it to state-of-the-art tools for transcript quantification. SpliceTrap demonstrated improved accuracy, robustness and reliability in quantifying exon-inclusion ratios.
Conclusions: SpliceTrap is a useful tool to study alternative splicing regulation, especially for accurate quantification of local exon-inclusion ratios from RNA-seq data.
Availability and Implementation: SpliceTrap can be implemented online through the CSH Galaxy server http://cancan.cshl.edu/splicetrap and is also available for download and installation at http://rulai.cshl.edu/splicetrap/.
Contact: michael.zhang@utdallas.edu
Supplementary Information: Supplementary data are available at Bioinformatics online.
doi:10.1093/bioinformatics/btr508
PMCID: PMC3198574  PMID: 21896509
13.  Directional DNA Methylation Changes and Complex Intermediate States Accompany Lineage Specificity in the Adult Hematopoietic Compartment 
Molecular cell  2011;44(1):17-28.
SUMMARY
DNA methylation has been implicated as an epigenetic component of mechanisms that stabilize cell-fate decisions. Here, we have characterized the methylomes of human female hematopoietic stem/progenitor cells (HSPCs) and mature cells from the myeloid and lymphoid lineages. Hypomethylated regions (HMRs) associated with lineage-specific genes were often methylated in the opposing lineage. In HSPCs, these sites tended to show intermediate, complex patterns that resolve to uniformity upon differentiation, by increased or decreased methylation. Promoter HMRs shared across diverse cell types typically display a constitutive core that expands and contracts in a lineage-specific manner to fine-tune the expression of associated genes. Many newly identified intergenic HMRs, both constitutive and lineage specific, were enriched for factor binding sites with an implied role in genome organization and regulation of gene expression, respectively. Overall, our studies represent an important reference data set and provide insights into directional changes in DNA methylation as cells adopt terminal fates.
doi:10.1016/j.molcel.2011.08.026
PMCID: PMC3412369  PMID: 21924933
14.  Sperm methylation profiles reveal features of epigenetic inheritance and evolution in primates 
Cell  2011;146(6):1029-1041.
Summary
During germ cell and preimplantation development, mammalian cells undergo nearly complete reprogramming of DNA methylation patterns. We profiled the methylomes of human and chimp sperm as a basis for comparison to methylation patterns of ES cells. While the majority of promoters escape methylation in both ES cells and sperm, the corresponding hypomethylated regions show substantial structural differences. Repeat elements are heavily methylated in both germ and somatic cells; however, retrotransposons from several subfamilies evade methylation more effectively during male germ cell development, while other subfamilies show the opposite trend. Comparing methylomes of human and chimp sperm revealed a subset of differentially methylated promoters and strikingly divergent methylation in retrotransposon subfamilies, with an evolutionary impact that is apparent in the underlying genomic sequence. Thus, the features that determine DNA methylation patterns differ between male germ cells and somatic cells, and elements of these features have diverged between humans and chimpanzees.
doi:10.1016/j.cell.2011.08.016
PMCID: PMC3205962  PMID: 21925323
15.  A Functional Phylogenomic View of the Seed Plants 
PLoS Genetics  2011;7(12):e1002411.
A novel result of the current research is the development and implementation of a unique functional phylogenomic approach that explores the genomic origins of seed plant diversification. We first use 22,833 sets of orthologs from the nuclear genomes of 101 genera across land plants to reconstruct their phylogenetic relationships. One of the more salient results is the resolution of some enigmatic relationships in seed plant phylogeny, such as the placement of Gnetales as sister to the rest of the gymnosperms. In using this novel phylogenomic approach, we were also able to identify overrepresented functional gene ontology categories in genes that provide positive branch support for major nodes prompting new hypotheses for genes associated with the diversification of angiosperms. For example, RNA interference (RNAi) has played a significant role in the divergence of monocots from other angiosperms, which has experimental support in Arabidopsis and rice. This analysis also implied that the second largest subunit of RNA polymerase IV and V (NRPD2) played a prominent role in the divergence of gymnosperms. This hypothesis is supported by the lack of 24nt siRNA in conifers, the maternal control of small RNA in the seeds of flowering plants, and the emergence of double fertilization in angiosperms. Our approach takes advantage of genomic data to define orthologs, reconstruct relationships, and narrow down candidate genes involved in plant evolution within a phylogenomic view of species' diversification.
Author Summary
Understanding the genetic and genomic basis of plant diversification has been a major goal of evolutionary biologists since Darwin first pondered his “abominable mystery,” the rapid diversification of the angiosperms in the fossil record. We develop and deploy a functional phylogenomic approach that helps identify genes and biological processes putatively involved in species diversification. We assembled a matrix of 22,833 orthologs from 150 species to reconstruct seed plant phylogenetic relationships and to identify gene sets with a unique evolutionary signal. Our analysis of overrepresented biological processes in these sets narrowed down possible genetic mechanisms underlying plant adaptation and diversification. The phylogenetic relationships we uncovered support the hypothesis that gnetophytes are closely related to the rest of the gymnosperms at the base of the living seed plants. We also found that genes involved in post-transcriptional silencing via RNA interference (RNAi)—increasingly important in understanding plant evolution—are significantly represented early in angiosperm and gymnosperm divergence, with an apparent loss of specific classes of small interfering RNAs (siRNA) in gymnosperms. Our functional phylogenomic approach can be applied to any taxa with available sequences to enhance our knowledge of the evolutionary processes underlying biodiversity in general.
doi:10.1371/journal.pgen.1002411
PMCID: PMC3240601  PMID: 22194700
16.  Establishing the baseline level of repetitive element expression in the human cortex 
BMC Genomics  2011;12:495.
Background
Although nearly half of the human genome is comprised of repetitive sequences, the expression profile of these elements remains largely uncharacterized. Recently developed high throughput sequencing technologies provide us with a powerful new set of tools to study repeat elements. Hence, we performed whole transcriptome sequencing to investigate the expression of repetitive elements in human frontal cortex using postmortem tissue obtained from the Stanley Medical Research Institute.
Results
We found a significant amount of reads from the human frontal cortex originate from repeat elements. We also noticed that Alu elements were expressed at levels higher than expected by random or background transcription. In contrast, L1 elements were expressed at lower than expected amounts.
Conclusions
Repetitive elements are expressed abundantly in the human brain. This expression pattern appears to be element specific and can not be explained by random or background transcription. These results demonstrate that our knowledge about repetitive elements is far from complete. Further characterization is required to determine the mechanism, the control, and the effects of repeat element expression.
doi:10.1186/1471-2164-12-495
PMCID: PMC3207997  PMID: 21985647
17.  A comparative analysis of exome capture 
Genome Biology  2011;12(9):R97.
Background
Human exome resequencing using commercial target capture kits has been and is being used for sequencing large numbers of individuals to search for variants associated with various human diseases. We rigorously evaluated the capabilities of two solution exome capture kits. These analyses help clarify the strengths and limitations of those data as well as systematically identify variables that should be considered in the use of those data.
Results
Each exome kit performed well at capturing the targets they were designed to capture, which mainly corresponds to the consensus coding sequences (CCDS) annotations of the human genome. In addition, based on their respective targets, each capture kit coupled with high coverage Illumina sequencing produced highly accurate nucleotide calls. However, other databases, such as the Reference Sequence collection (RefSeq), define the exome more broadly, and so not surprisingly, the exome kits did not capture these additional regions.
Conclusions
Commercial exome capture kits provide a very efficient way to sequence select areas of the genome at very high accuracy. Here we provide the data to help guide critical analyses of sequencing data derived from these products.
doi:10.1186/gb-2011-12-9-r97
PMCID: PMC3308060  PMID: 21958622
18.  Hybrid selection of discrete genomic intervals on custom-designed microarrays for massively parallel sequencing 
Nature protocols  2009;4(6):960-974.
Complementary techniques that deepen information content and minimize reagent costs are required to realize the full potential of massively parallel sequencing. Here, we describe a resequencing approach that directs focus to genomic regions of high interest by combining hybridization-based purification of multi-megabase regions with sequencing on the Illumina Genome Analyzer (GA). The capture matrix is created by a microarray on which probes can be programmed as desired to target any non-repeat portion of the genome, while the method requires only a basic familiarity with microarray hybridization. We present a detailed protocol suitable for 1–2 µg of input genomic DNA and highlight key design tips in which high specificity (>65% of reads stem from enriched exons) and high sensitivity (98% targeted base pair coverage) can be achieved. We have successfully applied this to the enrichment of coding regions, in both human and mouse, ranging from 0.5 to 4 Mb in length. From genomic DNA library production to base-called sequences, this procedure takes approximately 9–10 d inclusive of array captures and one Illumina flow cell run.
doi:10.1038/nprot.2009.68
PMCID: PMC2990409  PMID: 19478811
19.  Alta-Cyclic: a self-optimizing base caller for next-generation sequencing 
Nature methods  2008;5(8):679-682.
Next-generation sequencing is limited to short read lengths and by high error rates. We systematically analyzed sources of noise in the Illumina Genome Analyzer that contribute to these high error rates and developed a base caller, Alta-Cyclic, that uses machine learning to compensate for noise factors. Alta-Cyclic substantially improved the number of accurate reads for sequencing runs up to 78 bases and reduced systematic biases, facilitating confident identification of sequence variants.
doi:10.1038/nmeth.1230
PMCID: PMC2978646  PMID: 18604217
20.  Specialized piRNA Pathways Act in Germline and Somatic Tissues of the Drosophila Ovary 
Cell  2009;137(3):522-535.
SUMMARY
In Drosophila gonads, Piwi proteins and associated piRNAs collaborate with additional factors to form a small RNA-based immune system that silences mobile elements. Here, we analyzed nine Drosophila piRNA pathway mutants for their impacts on both small RNA populations and the subcellular localization patterns of Piwi proteins. We find that distinct piRNA pathways with differing components function in ovarian germ and somatic cells. In the soma, Piwi acts singularly with the conserved flamenco piRNA cluster to enforce silencing of retroviral elements that may propagate by infecting neighboring germ cells. In the germline, silencing programs encoded within piRNA clusters are optimized via a slicer-dependent amplification loop to suppress a broad spectrum of elements. The classes of transposons targeted by germline and somatic piRNA clusters, though not the precise elements, are conserved among Drosophilids, demonstrating that the architecture of piRNA clusters has coevolved with the transposons that they are tasked to control.
doi:10.1016/j.cell.2009.03.040
PMCID: PMC2882632  PMID: 19395010
21.  Detailed Analysis of a Contiguous 22-Mb Region of the Maize Genome 
PLoS Genetics  2009;5(11):e1000728.
Most of our understanding of plant genome structure and evolution has come from the careful annotation of small (e.g., 100 kb) sequenced genomic regions or from automated annotation of complete genome sequences. Here, we sequenced and carefully annotated a contiguous 22 Mb region of maize chromosome 4 using an improved pseudomolecule for annotation. The sequence segment was comprehensively ordered, oriented, and confirmed using the maize optical map. Nearly 84% of the sequence is composed of transposable elements (TEs) that are mostly nested within each other, of which most families are low-copy. We identified 544 gene models using multiple levels of evidence, as well as five miRNA genes. Gene fragments, many captured by TEs, are prevalent within this region. Elimination of gene redundancy from a tetraploid maize ancestor that originated a few million years ago is responsible in this region for most disruptions of synteny with sorghum and rice. Consistent with other sub-genomic analyses in maize, small RNA mapping showed that many small RNAs match TEs and that most TEs match small RNAs. These results, performed on ∼1% of the maize genome, demonstrate the feasibility of refining the B73 RefGen_v1 genome assembly by incorporating optical map, high-resolution genetic map, and comparative genomic data sets. Such improvements, along with those of gene and repeat annotation, will serve to promote future functional genomic and phylogenomic research in maize and other grasses.
Author Summary
Maize is a major cereal crop and key experimental system for eukaryotic biology. Previous investigations of the maize genome at the sequence level have primarily focused on analyses of genome survey sequences and BAC contigs. Here we used a comprehensive set of resources to construct an ordered and oriented 22-Mb sequence from chromosome 4 that represents 1% of the maize genome. Genome annotation revealed the presence of 544 genes that are interspersed with transposable elements (TEs), which occupy 83.8% of the sequence. Fifty-one genes were involved in 14 tandem gene clusters and most appear to have arisen after lineage divergence. TEs, especially helitrons, were found to contain gene fragments and were widely distributed in gene-rich regions. Large inversions and unequal gene deletion between the two homoeologous maize regions were the main contributors to synteny disruption among maize, sorghum, and rice. We also show that small RNAs are primarily associated with TEs across the region. Comparison of this ordered and oriented sequence with the corresponding uncurated region in the whole genome sequence of maize resulted in improvements in TE annotation that will ultimately enhance detection sensitivity and characterization of TEs. Doing so is likely to improve the specificity of gene annotations.
doi:10.1371/journal.pgen.1000728
PMCID: PMC2773423  PMID: 19936048
22.  Epigenetic Natural Variation in Arabidopsis thaliana 
PLoS Biology  2007;5(7):e174.
Cytosine methylation of repetitive sequences is widespread in plant genomes, occurring in both symmetric (CpG and CpNpG) as well as asymmetric sequence contexts. We used the methylation-dependent restriction enzyme McrBC to profile methylated DNA using tiling microarrays of Arabidopsis Chromosome 4 in two distinct ecotypes, Columbia and Landsberg erecta. We also used comparative genome hybridization to profile copy number polymorphisms. Repeated sequences and transposable elements (TEs), especially long terminal repeat retrotransposons, are densely methylated, but one third of genes also have low but detectable methylation in their transcribed regions. While TEs are almost always methylated, genic methylation is highly polymorphic, with half of all methylated genes being methylated in only one of the two ecotypes. A survey of loci in 96 Arabidopsis accessions revealed a similar degree of methylation polymorphism. Within-gene methylation is heritable, but is lost at a high frequency in segregating F2 families. Promoter methylation is rare, and gene expression is not generally affected by differences in DNA methylation. Small interfering RNA are preferentially associated with methylated TEs, but not with methylated genes, indicating that most genic methylation is not guided by small interfering RNA. This may account for the instability of gene methylation, if occasional failure of maintenance methylation cannot be restored by other means.
Author Summary
In plants and animals, many DNA sequences are modified by the addition of methyl groups, but the principles governing methylation patterns are not well understood. In Arabidopsis, we show that repetitive sequences, derived from mobile (transposable) elements, are densely methylated throughout their length, while about one third of all protein-coding genes are internally methylated. Methylated transposons are silent, homologous to small interfering RNA, and coated with histone H3 dimethylated on lysine-9. In contrast, methylated coding-sequence genes are highly expressed, do not have corresponding small RNAs, and are coated with histone H3 dimethylated on lysine-4. Comparing two different ecotypes of Arabidopsis, we find that transposons are twice as likely as genes to have suffered insertion and deletion, although gene deletion is surprisingly prevalent. While the pattern of transposon methylation is conserved between ecotypes, protein-coding gene methylation is polymorphic so that only half of all gene methylation on any one chromosome is shared between natural accessions collected from around the world.
Two ecotypes ofArabidopis show different patterns of DNA methylation, which is heritable. Interestingly, differences in DNA methylation are not reflected in differences in gene expression.
doi:10.1371/journal.pbio.0050174
PMCID: PMC1892575  PMID: 17579518
23.  On the importance of being finished 
Genome Biology  2002;3(10):comment2010.1-comment2010.4.
The publication of an increasing number of draft genome sequences presents problems that will only be resolved by improved search tools and by complete finishing of the sequences - and their deposition in publicly accessible databases.
The publication of an increasing number of draft genome sequences presents problems that will only be resolved by improved search tools and by complete finishing of the sequences - and their deposition in publicly accessible databases.
doi:10.1186/gb-2002-3-10-comment2010
PMCID: PMC244905  PMID: 12372139
24.  Sorghum Genome Sequencing by Methylation Filtration 
PLoS Biology  2005;3(1):e13.
Sorghum bicolor is a close relative of maize and is a staple crop in Africa and much of the developing world because of its superior tolerance of arid growth conditions. We have generated sequence from the hypomethylated portion of the sorghum genome by applying methylation filtration (MF) technology. The evidence suggests that 96% of the genes have been sequence tagged, with an average coverage of 65% across their length. Remarkably, this level of gene discovery was accomplished after generating a raw coverage of less than 300 megabases of the 735-megabase genome. MF preferentially captures exons and introns, promoters, microRNAs, and simple sequence repeats, and minimizes interspersed repeats, thus providing a robust view of the functional parts of the genome. The sorghum MF sequence set is beneficial to research on sorghum and is also a powerful resource for comparative genomics among the grasses and across the entire plant kingdom. Thousands of hypothetical gene predictions in rice and Arabidopsis are supported by the sorghum dataset, and genomic similarities highlight evolutionarily conserved regions that will lead to a better understanding of rice and Arabidopsis.
Methylation filtration makes practical the sequencing of large genomes, such as those found in sorghum, by preferentially capturing functionally relevant sequences
doi:10.1371/journal.pbio.0030013
PMCID: PMC539327  PMID: 15660154

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