Harmonizing the forensic nomenclature for STR loci D6S474 and DYS612.

The autosomal STR D6S474 and the Y-chromosomal STR DYS612 have been reported in multiple ways in the forensic literature, with differences in both the bracketed repeat structures and counting of numerical length-based capillary electrophoresis (CE) alleles. These issues often come to light when STR loci are introduced in commercial assays and results compared with historical publications of allele frequency data, or multiple assays are characterized with reference materials. We review the forensic literature and other relevant information, and provide suggestions for the future treatment of each STR.

US Population Data for 94 Identity-Informative SNP Loci.

The US National Institute of Standards and Technology (NIST) analyzed a set of 1036 samples representing four major US population groups (African American, Asian American, Caucasian, and Hispanic) with 94 single nucleotide polymorphisms (SNPs) used for individual identification (iiSNPs). The compact size of iiSNP amplicons compared to short tandem repeat (STR) markers increases the likelihood of successful amplification with degraded DNA samples. Allele frequencies and relevant forensic statistics were calculated for each population group as well as the aggregate population sample. Examination of sequence data in the regions flanking the targeted SNPs identified additional variants, which can be combined with the target SNPs to form microhaplotypes (multiple phased SNPs within a short-read sequence). Comparison of iiSNP performance with and without flanking SNP variation identified four amplicons containing microhaplotypes with observed heterozygosity increases of greater than 15% over the targeted SNP alone. For this set of 1036 samples, comparison of average match probabilities from iiSNPs with the 20 CODIS core STR markers yielded an estimate of 1.7 × 10-38 for iiSNPs (assuming independence between all 94 SNPs), which was four orders of magnitude lower (more discriminating) than STRs where internal sequence variation was considered, and 10 orders of magnitude lower than STRs using established capillary electrophoresis length-based genotypes.

Sequence-based allelic variations and frequencies for 22 autosomal STR loci in the Lebanese population.

This is the first study that characterizes the sequence-based allelic variations of 22 autosomal Short Tandem Repeat (aSTR) loci in a population dataset collected from Lebanon. Genomic DNA extracts from 195 unrelated Lebanese individuals were amplified with PowerSeq 46GY System Prototype. Targeted amplicons were subjected to DNA library preparation and sequenced on the Verogen MiSeq FGx Sequencing System. Raw FASTQ data files were processed by STRait Razor v3. Sequence strings were annotated according to the considerations of the DNA Commission of the International Society for Forensic Genetics (ISFG) and tabulated herein with their respective allelic frequencies and GeneBank accession and version numbers. The sequenced Lebanese dataset resulted in 429 distinct allelic sequences as compared to the 236 alleles identified by length only. The increase in the number of alleles was observed at 18 out of 22 aSTR loci and was attributed to the sequence variations residing in both the STR repeat motifs and flanking regions. The study uncovered 25 novel aSTR allelic sequences across 12 loci for which GenBank records did not previously exist in the STRSeq BioProject, PRJNA380127. For a concordance check, the length-based allelic calls derived from the full sequences were compared to those genotyped using capillary electrophoresis (CE) methods. Population genetic parameters relevant to the evaluation of forensic DNA evidence were assessed for the sequence-based data and compared to the parameters generated from the length-based information. Using the sequence-based data, Analysis of MOlecular VAriance (AMOVA), genetic distances, and population genetic structure were evaluated for 1231 individuals sampled from the Lebanese and four U.S. populations (African American, Asian, Caucasian, and Hispanic). The results were tabulated and visualized in a population tree, multidimensional scaling scatter plots, and bar plots. This newly established sequence-based database for the Lebanese population can be beneficial for extending NGS applicability to casework or paternity testing and assessing the strength of evidence for NGS-STR profiles. The described novel sequence variants at certain loci can further help in the effort to characterize the sequence diversity of STR markers from different populations around the world.

A multi-dimensional evaluation of the 'NIST 1032' sample set across four forensic Y-STR multiplexes.

This manuscript reports Y-chromosomal short tandem repeat (Y-STR) haplotypes for 1032 male U.S. population samples across 30 Y-STR loci characterized by three capillary electrophoresis (CE) length-based kits (PowerPlex Y23 System, Yfiler Plus PCR Amplification Kit, and Investigator Argus Y-28 QS Kit) and one sequence-based kit (ForenSeq DNA Signature Prep Kit): DYF387S1, DYS19, DYS385 a/b, DYS389I, DYS389II, DYS390, DYS391, DYS392, DYS393, DYS437, DYS438, DYS439, DYS448, DYS449, DYS456, DYS458, DYS460, DYS481, DYS505, DYS518, DYS522, DYS533, DYS549, DYS570, DYS576, DYS612, DYS627, DYS635, DYS643, and Y-GATA-H4. The length-based Y-STR haplotypes include six loci that are not reported in the sequence-based kit (DYS393, DYS449, DYS456, DYS458, DYS518, and DYS627), whereas three loci included in the sequence-based kit are not present in length-based kits (DYS505, DYS522, and DYS612). For the latter, a custom multiplex was used to generate CE length-based data, allowing 1032 samples to be evaluated for concordance across the 30 Y-STR loci included in these four commercial Y-STR typing kits. Discordances between typing methods were analyzed further to assess underlying causes such as primer binding site mutations and flanking region insertions/deletions. Allele-level frequency and statistical information is provided for sequenced loci, excluding the multi-copy loci DYF387S1 and DYS385 a/b, for which locus-specific haplotype-level frequencies are provided instead. The resulting data reveals the degree of information gained through sequencing: 88% of sequenced Y-STR loci contain additional sequence-based alleles compared to length-based data, with the DYS389II locus containing the most additional alleles (51) observed by sequencing. Despite these allelic increases, only minimal improvement was observed in haplotype resolution by sequence, with all four commercial kits providing a similar ability to differentiate length-based haplotypes in this sample set. Finally, a subset of 369 male samples were compared to their corresponding additionally sequenced father samples, revealing the sequence basis for the 50 length-based changes observed, and no additional sequence-based mutations. GenBank accession numbers are reported for each unique sequence, and associated records are available in the STRSeq Y-Chromosomal STR Loci National Center for Biotechnology Information (NCBI) BioProject, accession PRJNA380347. Haplotype data is updated in the Y-STR Haplotype Reference Database (YHRD) for the 'NIST 1032' data set to now achieve the level of maximal haplotype of YHRD. All supplementary files including revisions to previously published Y-STR data are available in the NIST Public Data Repository: U.S. population data for human identification markers, DOI 10.18434/t4/1500024.

Understanding the characteristics of sequence-based single-source DNA profiles.

The sequencing of STR markers provides additional information present in the underlying sequence variation that is typically masked by traditional fragment-based genotyping. However, the interpretation of STR profiles generated by targeted sequencing methods are susceptible to the same factors encountered in profiles processed through capillary gel electrophoresis. These factors include stochastic variation, noise, stutter artifacts, heterozygote imbalance, and allelic drop-out/in. Our goal is to characterize and understand how these behave in targeted sequence datasets. Here, we developed a framework using statistical tools to systematically interpret the characteristics of single-source DNA profiles generated by targeted sequencing. Sensitivity studies were performed using known single-source samples amplified with the PowerSeq 46GY System Prototype with varying DNA target masses ranging from 15 pg to 500 pg. The STR loci were subjected to DNA library preparation using two commercially available library kits and sequenced on the Illumina MiSeq platform. Raw FASTQ data files were analyzed in STRait Razor v2.0 without applying any thresholds (at a coverage ≥ 1). We investigated the effect of library normalization on average locus coverage and studied methods for setting analytical and zygosity thresholds. All the data were analyzed per DNA quantity as well as investigated per method. Analyses presented can be applied to sequence data generated by similar targeted sequencing panels and/or NGS platforms.

Report from the STRAND Working Group on the 2019 STR sequence nomenclature meeting.

This report summarizes topics discussed at the STR sequence nomenclature meeting hosted by the STRAND Working Group in April 2019. Invited attendees for this meeting included researchers known-to-us to be developing STR sequence-based nomenclature schemata, scientific representatives from vendors developing STR sequence bioinformatic methods, DNA intelligence database curators, and academic experts in STR genomics. The goal of this meeting was to provide a forum for individuals developing nomenclature schemata to present and discuss their ideas, encouraging mutual awareness, identification of differences in approaches, opposing aspects, and opportunities for parallelization while some approaches are still under development.

Sequence-based U.S. population data for 27 autosomal STR loci.

This manuscript reports Short Tandem Repeat (STR) sequence-based allele frequencies for 1036 samples across 27 autosomal STR loci: D1S1656, TPOX, D2S441, D2S1338, D3S1358, D4S2408, FGA, D5S818, CSF1PO, D6S1043, D7S820, D8S1179, D9S1122, D10S1248, TH01, vWA, D12S391, D13S317, Penta E, D16S539, D17S1301, D18S51, D19S433, D20S482, D21S11, Penta D, and D22S1045. Sequence data were analyzed by two bioinformatic pipelines and all samples have been evaluated for concordance with alleles derived from CE-based analysis at all loci. Each reported sequence includes high-quality flanking sequence and is properly formatted according to the most recent guidance of the International Society for Forensic Genetics. In addition, GenBank accession numbers are reported for each sequence, and associated records are available in the STRSeq BioProject (https://www.ncbi.nlm.nih.gov/bioproject/380127). The D3S1358 locus demonstrates the greatest average increase in heterozygosity across populations (approximately 10 percentage points). Loci demonstrating average increase in heterozygosity from 10 to 5 percentage points include (in descending order) D9S1122, D13S317, D8S1179, D21S11, D5S818, D12S391, and D2S441. The remaining 19 loci each demonstrate less than 5 percentage point increase in average heterozygosity. Discussion includes the utility of this data in understanding traditional CE results, such as informing stutter models and understanding migration challenges, and considerations for population sampling strategies in light of the marked increase in rare alleles for several of the sequence-based STR loci. This NIST 1036 data set is expected to support the implementation of STR sequencing forensic casework by providing high-confidence sequence-based allele frequencies for the same sample set which are already the basis for population statistics in many U.S. forensic laboratories.

Sequence-based US population data for the SE33 locus.

A set of 1036 U.S. Population Samples were sequenced using the Illumina ForenSeq DNA Signature Prep Kit. This sample set has been highly characterized using a variety of marker systems for human identification. The FASTQ files obtained from a ForenSeq DNA Signature Prep Kit experiment include several STR loci that are not reported in the associated software. These include SE33, DXS8377, DXS10148, DYS456, and DYS461. The sequence variation within the autosomal STR marker SE33 was evaluated using a customized bioinformatic approach to identify and characterize the locus in the 1036 data set. The analysis identified 53 unique alleles by length and 264 by sequence. An additional 10 alleles were detected when selected extended flanking regions were examined to resolve discordances. Allele frequencies and SE33 sequence motif patterns are reported for the 1036 data set. The comparison of numerical allele calls derived from sequence data to the allele calls obtained from commercial capillary electrophoresis-based STR typing kits resulted in 100% concordance, after manual data review and confirmation sequencing of three flanking region deletions. The analysis of this data set involved significant manual sequence curation and information support from length-based genotypes to ensure high confidence in the sequence-based allele calls. The challenges of interpreting the sequence data for SE33 consisted of high sequence noise, allele-size dependent variance in coverage, and heterozygote imbalance. As allele length increased, sequence depth of coverage and quality decreased at the terminal end. Accordingly, heterozygous genotype imbalance increased in proportion to increased distance between alleles.

STRSeq: A catalog of sequence diversity at human identification Short Tandem Repeat loci.

The STR Sequencing Project (STRSeq) was initiated to facilitate the description of sequence-based alleles at the Short Tandem Repeat (STR) loci targeted in human identification assays. This international collaborative effort, which has been endorsed by the ISFG DNA Commission, provides a framework for communication among laboratories. The initial data used to populate the project are the aggregate alleles observed in targeted sequencing studies across four laboratories: National Institute of Standards and Technology (N=1786), Kings College London (N=1043), University of North Texas Health Sciences Center (N=839), and University of Santiago de Compostela (N=944), for a total of 4612 individuals. STRSeq data are maintained as GenBank records at the U.S. National Center for Biotechnology Information (NCBI), which participates in a daily data exchange with the DNA DataBank of Japan (DDBJ) and the European Nucleotide Archive (ENA). Each GenBank record contains the observed sequence of a STR region, annotation ("bracketing") of the repeat region and flanking region polymorphisms, information regarding the sequencing assay and data quality, and backward compatible length-based allele designation. STRSeq GenBank records are organized within a BioProject at NCBI (https://www.ncbi.nlm.nih.gov/bioproject/380127), which is sub-divided into: commonly used autosomal STRs, alternate autosomal STRs, Y-chromosomal STRs, and X-chromosomal STRs. Each of these categories is further divided into locus-specific BioProjects. The BioProject hierarchy facilitates access to the GenBank records by browsing, BLAST searching, or ftp download. Future plans include user interface tools at strseq.nist.gov, a pathway for submission of additional allele records by laboratories performing population sample sequencing and interaction with the STRidER web portal for quality control (http://strider.online).

Characterization of NIST human mitochondrial DNA SRM-2392 and SRM-2392-I standard reference materials by next generation sequencing.

Standard Reference Materials SRM 2392 and 2392-I are intended to provide quality control when amplifying and sequencing human mitochondrial genome sequences. The National Institute of Standards and Technology (NIST) offers these SRMs to laboratories performing DNA-based forensic human identification, molecular diagnosis of mitochondrial diseases, mutation detection, evolutionary anthropology, and genetic genealogy. The entire mtGenome (∼16569bp) of SRM 2392 and 2392-I have previously been characterized at NIST by Sanger sequencing. Herein, we used the sensitivity, specificity, and accuracy offered by next generation sequencing (NGS) to: (1) re-sequence the certified values of the SRM 2392 and 2392-I; (2) confirm Sanger data with a high coverage new sequencing technology; (3) detect lower level heteroplasmies (<20%); and thus (4) support mitochondrial sequencing communities in the adoption of NGS methods. To obtain a consensus sequence for the SRMs as well as identify and control any bias, sequencing was performed using two NGS platforms and data was analyzed using different bioinformatics pipelines. Our results confirm five low level heteroplasmy sites that were not previously observed with Sanger sequencing: three sites in the GM09947A template in SRM 2392 and two sites in the HL-60 template in SRM 2392-I.

Single assay for simultaneous detection and differential identification of human and avian influenza virus types, subtypes, and emergent variants.

For more than four decades the cause of most type A influenza virus infections of humans has been attributed to only two viral subtypes, A/H1N1 or A/H3N2. In contrast, avian and other vertebrate species are a reservoir of type A influenza virus genome diversity, hosting strains representing at least 120 of 144 combinations of 16 viral hemagglutinin and 9 viral neuraminidase subtypes. Viral genome segment reassortments and mutations emerging within this reservoir may spawn new influenza virus strains as imminent epidemic or pandemic threats to human health and poultry production. Traditional methods to detect and differentiate influenza virus subtypes are either time-consuming and labor-intensive (culture-based) or remarkably insensitive (antibody-based). Molecular diagnostic assays based upon reverse transcriptase-polymerase chain reaction (RT-PCR) have short assay cycle time, and high analytical sensitivity and specificity. However, none of these diagnostic tests determine viral gene nucleotide sequences to distinguish strains and variants of a detected pathogen from one specimen to the next. Decision-quality, strain- and variant-specific pathogen gene sequence information may be critical for public health, infection control, surveillance, epidemiology, or medical/veterinary treatment planning. The Resequencing Pathogen Microarray (RPM-Flu) is a robust, highly multiplexed and target gene sequencing-based alternative to both traditional culture- or biomarker-based diagnostic tests. RPM-Flu is a single, simultaneous differential diagnostic assay for all subtype combinations of type A influenza viruses and for 30 other viral and bacterial pathogens that may cause influenza-like illness. These other pathogen targets of RPM-Flu may co-infect and compound the morbidity and/or mortality of patients with influenza. The informative specificity of a single RPM-Flu test represents specimen-specific viral gene sequences as determinants of virus type, A/HN subtype, virulence, host-range, and resistance to antiviral agents.

Floret-specific differences in gene expression and support for the hypothesis that tapetal degeneration of Zea mays L. occurs via programmed cell death.

The maize (Zea mays) spikelet consists of two florets, each of which contains three developmentally synchronized anthers. Morphologically, the anthers in the upper and lower florets proceed through apparently similar developmental programs. To test for global differences in gene expression and to identify genes that are coordinately regulated during maize anther development, RNA samples isolated from upper and lower floret anthers at six developmental stages were hybridized to cDNA microarrays. Approximately 9% of the tested genes exhibited statistically significant differences in expression between anthers in the upper and lower florets. This finding indicates that several basic biological processes are differentially regulated between upper and lower floret anthers, including metabolism, protein synthesis and signal transduction. Genes that are coordinately regulated across anther development were identified via cluster analysis. Analysis of these results identified stage-specific, early in development, late in development and bi-phasic expression profiles. Quantitative RT-PCR analysis revealed that four genes whose homologs in other plant species are involved in programmed cell death are up-regulated just prior to the time the tapetum begins to visibly degenerate (i.e., the mid-microspore stage). This finding supports the hypothesis that developmentally normal tapetal degeneration occurs via programmed cell death.

Transcriptomic and proteomic analyses of pericycle cells of the maize primary root.

Each plant cell type expresses a unique transcriptome and proteome at different stages of differentiation dependent on its developmental fate. This study compared gene expression and protein accumulation in cell-cycle-competent primary root pericycle cells of maize (Zea mays) prior to their first division and lateral root initiation. These are the only root cells that maintain the competence to divide after they leave the meristematic zone. Pericycle cells of the inbred line B73 were isolated via laser capture microdissection. Microarray experiments identified 32 genes preferentially expressed in pericycle versus all other root cells that have left the apical meristem; selective subtractive hybridization identified seven genes preferentially expressed in pericycle versus central cylinder cells of the same root region. Transcription and protein synthesis represented the most abundant functional categories among these pericycle-specific genes. Moreover, 701 expressed sequence tags (ESTs) were generated from pericycle and central cylinder cells. Among those, transcripts related to protein synthesis and cell fate were significantly enriched in pericycle versus nonpericycle cells. In addition, 77 EST clusters not previously identified in maize ESTs or genomic databases were identified. Finally, among the most abundant soluble pericycle proteins separated via two-dimensional electrophoresis, 20 proteins were identified via electrospray ionization-tandem mass spectrometry, thus defining a reference dataset of the maize pericycle proteome. Among those, two proteins were preferentially expressed in the pericycle. In summary, these pericycle-specific gene expression experiments define the distinct molecular events during the specification of cell-cycle-competent pericycle cells prior to their first division and demonstrate that pericycle specification and lateral root initiation might be controlled by a different set of genes.

Global gene expression analysis of the shoot apical meristem of maize (Zea mays L.).

All above-ground plant organs are derived from shoot apical meristems (SAMs). Global analyses of gene expression were conducted on maize (Zea mays L.) SAMs to identify genes preferentially expressed in the SAM. The SAMs were collected from 14-day-old B73 seedlings via laser capture microdissection (LCM). The RNA samples extracted from LCM-collected SAMs and from seedlings were hybridized to microarrays spotted with 37 660 maize cDNAs. Approximately 30% (10 816) of these cDNAs were prepared as part of this study from manually dissected B73 maize apices. Over 5000 expressed sequence tags (ESTs) (about 13% of the total) were differentially expressed (P < 0.0001) between SAMs and seedlings. Of these, 2783 and 2248 ESTs were up- and down-regulated in the SAM, respectively. The expression in the SAM of several of the differentially expressed ESTs was validated via quantitative RT-PCR and/or in situ hybridization. The up-regulated ESTs included many regulatory genes including transcription factors, chromatin remodeling factors and components of the gene-silencing machinery, as well as about 900 genes with unknown functions. Surprisingly, transcripts that hybridized to 62 retrotransposon-related cDNAs were also substantially up-regulated in the SAM. Complementary DNAs derived from the LCM-collected SAMs were sequenced to identify additional genes that are expressed in the SAM. This generated around 550 000 ESTs (454-SAM ESTs) from two genotypes. Consistent with the microarray results, approximately 14% of the 454-SAM ESTs from B73 were retrotransposon-related. Possible roles of genes that are preferentially expressed in the SAM are discussed.

Scanning microarrays at multiple intensities enhances discovery of differentially expressed genes.

MOTIVATION: Scanning parameters are often overlooked when optimizing microarray experiments. A scanning approach that extends the dynamic data range by acquiring multiple scans of different intensities has been developed. RESULTS: Data from each of three scan intensities (low, medium, high) were analyzed separately using multiple scan and linear regression approaches to identify and compare the sets of genes that exhibit statistically significant differential expression. In the multiple scan approach only one-third of the differentially expressed genes were shared among the three intensities, and each scan intensity identified unique sets of differentially expressed genes. The set of differentially expressed genes from any one scan amounted to < 70% of the total number of genes identified in at least one scan. The average signal intensity of genes that exhibited statistically significant changes in expression was highest for the low-intensity scan and lowest for the high-intensity scan, suggesting that low-intensity scans may be best for detecting expression differences in high-signal genes, while high-intensity scans may be best for detecting expression differences in low-signal genes. Comparison of the differentially expressed genes identified in the multiple scan and linear regression approaches revealed that the multiple scan approach effectively identifies a subset of statistically significant genes that linear regression approach is unable to identify. Quantitative RT-PCR (qRT-PCR) tests demonstrated that statistically significant differences identified at all three scan intensities can be verified. AVAILABILITY: The data presented can be viewed at http://www.ncbi.nlm.nih.gov/geo/ under GEO accession no. GSE3017.

All possible modes of gene action are observed in a global comparison of gene expression in a maize F1 hybrid and its inbred parents.

Heterosis is the phenomenon whereby the progeny of particular inbred lines have enhanced agronomic performance relative to both parents. Although several hypotheses have been proposed to explain this fundamental biological phenomenon, the responsible molecular mechanisms have not been determined. The maize inbred lines B73 and Mo17 produce a heterotic F1 hybrid. Global patterns of gene expression were compared in seedlings of these three genotypes by using a microarray that contains 13,999 cDNAs. Using an estimated 15% false discovery rate as a cutoff, 1,367 ESTs (9.8%) were identified as being significantly differentially expressed among genotypes. All possible modes of gene action were observed, including additivity, high- and low-parent dominance, underdominance, and overdominance. The largest proportion of the ESTs (78%; 1,062 of 1,367) exhibited expression patterns that are not statistically distinguishable from additivity. Even so, 22% of the differentially regulated ESTs exhibited nonadditive modes of gene expression. Classified on the basis of significant pairwise comparisons of genotype means, 181 of these 305 nonadditive ESTs exhibited high-parent dominance and 23 exhibited low-parent dominance. In addition, 44 ESTs exhibited underdominance or overdominance. These findings are consistent with the hypothesis that multiple molecular mechanisms, including overdominance, contribute to heterosis.

Isolation, characterization, and pericycle-specific transcriptome analyses of the novel maize lateral and seminal root initiation mutant rum1.

The monogenic recessive maize (Zea mays) mutant rootless with undetectable meristems 1 (rum1) is deficient in the initiation of the embryonic seminal roots and the postembryonic lateral roots at the primary root. Lateral root initiation at the shoot-borne roots and development of the aerial parts of the mutant rum1 are not affected. The mutant rum1 displays severely reduced auxin transport in the primary root and a delayed gravitropic response. Exogenously applied auxin does not induce lateral roots in the primary root of rum1. Lateral roots are initiated in a specific cell type, the pericycle. Cell-type-specific transcriptome profiling of the primary root pericycle 64 h after germination, thus before lateral root initiation, via a combination of laser capture microdissection and subsequent microarray analyses of 12k maize microarray chips revealed 90 genes preferentially expressed in the wild-type pericycle and 73 genes preferentially expressed in the rum1 pericycle (fold change >2; P-value <0.01; estimated false discovery rate of 13.8%). Among the 51 annotated genes predominately expressed in the wild-type pericycle, 19 genes are involved in signal transduction, transcription, and the cell cycle. This analysis defines an array of genes that is active before lateral root initiation and will contribute to the identification of checkpoints involved in lateral root formation downstream of rum1.

Evaluation of five ab initio gene prediction programs for the discovery of maize genes.

Five ab initio programs (FGENESH, GeneMark.hmm, GENSCAN, GlimmerR and Grail) were evaluated for their accuracy in predicting maize genes. Two of these programs, GeneMark.hmm and GENSCAN had been trained for maize; FGENESH had been trained for monocots (including maize), and the others had been trained for rice or Arabidopsis. Initial evaluations were conducted using eight maize genes (gl8a, pdc2, pdc3, rf2c, rf2d, rf2e1, rth1, and rth3) of which the sequences were not released to the public prior to conducting this evaluation. The significant advantage of this data set for this evaluation is that these genes could not have been included in the training sets of the prediction programs. FGENESH yielded the most accurate and GeneMark.hmm the second most accurate predictions. The five programs were used in conjunction with RT-PCR to identify and establish the structures of two new genes in the a1-sh2 interval of the maize genome. FGENESH, GeneMark.hmm and GENSCAN were tested on a larger data set consisting of maize assembled genomic islands (MAGIs) that had been aligned to ESTs. FGENESH, GeneMark.hmm and GENSCAN correctly predicted gene models in 773, 625, and 371 MAGIs, respectively, out of the 1353 MAGIs that comprise data set 2.

Laser-capture microdissection, a tool for the global analysis of gene expression in specific plant cell types: identification of genes expressed differentially in epidermal cells or vascular tissues of maize.

Laser-capture microdissection (LCM) allows for the one-step procurement of large homogeneous populations of cells from tissue sections. In mammals, LCM has been used to conduct cDNA microarray and proteomics studies on specific cell types. However, LCM has not been applied to plant cells, most likely because plant cell walls make it difficult to separate target cells from surrounding cells and because ice crystals can form in the air spaces between cells when preparing frozen sections. By fixing tissues, using a cryoprotectant before freezing, and using an adhesive-coated slide system, it was possible to capture large numbers (>10,000) of epidermal cells and vascular tissues (vascular bundles and bundle sheath cells) from ethanol:acetic acid-fixed coleoptiles of maize. RNA extracted from these cells was amplified with T7 RNA polymerase and used to hybridize a microarray containing approximately 8800 maize cDNAs. Approximately 250 of these were expressed preferentially in epidermal cells or vascular tissues. These results demonstrate that the combination of LCM and microarrays makes it feasible to conduct high-resolution global gene expression analyses of plants. This approach has the potential to enhance our understanding of diverse plant cell type-specific biological processes.