Massively parallel sequencing instruments enable rapid and inexpensive DNA sequence data production. Because these instruments are new, their data require characterization with respect to accuracy and utility. To address this, we sequenced a Caernohabditis elegans N2 Bristol strain isolate using the Solexa Sequence Analyzer, and compared the reads to the reference genome to characterize the data and to evaluate coverage and representation. Massively parallel sequencing facilitates strain-to-reference comparison for genome-wide sequence variant discovery. Owing to the short-read-length sequences produced, we developed a revised approach to determine the regions of the genome to which short reads could be uniquely mapped. We then aligned Solexa reads from C. elegans strain CB4858 to the reference, and screened for single-nucleotide polymorphisms (SNPs) and small indels. This study demonstrates the utility of massively parallel short read sequencing for whole genome resequencing and for accurate discovery of genome-wide polymorphisms.
Forward genetic mutational studies, adaptive evolution, and phenotypic screening are powerful tools for creating new variant organisms with desirable traits. However, mutations generated in the process cannot be easily identified with traditional genetic tools. We show that new high-throughput, massively parallel sequencing technologies can completely and accurately characterize a mutant genome relative to a previously sequenced parental (reference) strain. We studied a mutant strain of Pichia stipitis, a yeast capable of converting xylose to ethanol. This unusually efficient mutant strain was developed through repeated rounds of chemical mutagenesis, strain selection, transformation, and genetic manipulation over a period of seven years. We resequenced this strain on three different sequencing platforms. Surprisingly, we found fewer than a dozen mutations in open reading frames. All three sequencing technologies were able to identify each single nucleotide mutation given at least 10-15-fold nominal sequence coverage. Our results show that detecting mutations in evolved and engineered organisms is rapid and cost-effective at the whole-genome level using new sequencing technologies. Identification of specific mutations in strains with altered phenotypes will add insight into specific gene functions and guide further metabolic engineering efforts.[Supplemental material is available online at www.genome.org. Complete data sets are available at the NCBI Short Read Archive under accession no. SRA 001158 (ftp://ftp.ncbi.nih.gov/pub/TraceDB/ShortRead).]Pichia stipitis (Pignal) is a haploid yeast related to endosymbionts of beetles that degrade rotting wood (Suh et al. 2003). It is an important organism for bioenergy production from lignocellulosic materials because of its high capacity to ferment xylose and cellobiose to ethanol (Parekh et al. 1988). We previously sequenced the reference strain, Pichia stipitis CBS-6054, resulting in a completely characterized genome of eight chromosomes totaling 15.4 Mb of sequence (Jeffries et al. 2007). This strain has been subjected to chemical mutagenesis, phenotypic selection, genetic engineering, and adaptive evolution in order to develop strains improved for ethanol production. Chemical mutagenesis and selection resulted in small improvements in ethanol production attributable in part to carbon catabolite derepression (Supplemental Fig. 1; Methods). Disruption of CYC1 (cyctochrome c, isoform 1) to create strain Shi21 increased the specific ethanol production rate by 50% and the ethanol yield by 10%; however, the nature of additional mutational events leading to this phenotype was uncharacterized.Traditional methods for identifying mutations are laborand time-intensive, so we tested the ability of next-generation sequencing technologies to determine the differences in this improved strain's entire genome relative to the reference strain. We generated high-coverage, whole-genome data sets using single fragment end reads from three next-generation sequencing platforms: 454 Life Sc...
Previously reported applications of the 454 Life Sciences pyrosequencing technology have relied on deep sequence coverage for accurate polymorphism discovery because of frequent insertion and deletion sequence errors. Here we report a new base calling program, Pyrobayes, for pyrosequencing reads. Pyrobayes permits accurate single-nucleotide polymorphism (SNP) calling in resequencing applications, even in shallow read coverage, primarily because it produces more confident base calls than the native base calling program.
Klinefelter syndrome (KS) affects about 1 in 900 males due to an extra X chromosome. Although there are no obvious physical features associated with childhood KS, many boys demonstrate a cognitive deficit in verbal processing. The first section of this article integrates the extant literature on intelligence and achievement outcomes in boys with KS. The second section presents our findings from a 20-year study involving one of the largest unselected cohorts of boys with KS. We followed 36 boys with KS and 33 sibling controls from 6 until 20 years of age. Boys with KS are shown to demonstrate a verbal cognitive deficit and significant underachievement in reading and spelling, as well as in arithmetic. These problems, which are evident from early school years, increase with age such that by late adolescence, boys with KS are four to five grade levels behind. In addition, we also found that they were most likely to have a generalized type of learning disability, with very few boys indicating a pure reading or pure arithmetic problem. They also showed deficits in written language skills and acquisition of knowledge-based subject material were also problematic. Despite significant underachievement and frequent grade failure, many boys with KS had completed high school, and a few were also pursuing postsecondary educations. The discussion section examines how their language-based disability affects comprehension and learning, leading to underachievement.
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