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Because Kemp’s ridley sea turtles (Lepidochelys kempii) are critically endangered and closely related to the vulnerable olive ridleys (L. olivacea), it is essential for forensic investigations and conservation efforts to distinguish these species when only skeletal elements remain. DNA extraction and analysis by DNA sequencing of genetic markers is the only method to determine species identity reliably, yet these methods are significantly compromised when DNA becomes degraded. To evaluate the role that time and environment play in obtaining high-quality DNA sequencing data, we placed skeletal elements of a terrestrial mammal (Bos taurus) and L. kempii in a supratidal and subtidal environment for 3 years. Bi-annual sampling revealed that after 3 years, mitochondrial DNA (mtDNA) consistently identified each species from each environment. Our results show that mtDNA recovery from bone and identification for Kemp’s ridley sea turtles was possible up to 3 years in both environments. All sequencing data obtained was accurate and robust, but DNA sequencing results were not consistent after 664-days of exposure. Our findings led us to conclude that if sufficient DNA is extracted from bone samples, then high-quality sequence data can be obtained, and the resulting sequence data accurately reflects the reference sequence for the given gene marker. This study provides evidence that DNA can be extracted and analyzed from challenging biological substrates, like bone, when these substrates are exposed to seasonally dynamic maritime environmental conditions for up to 3-years.

Using quantitative PCR analysis and DNA sequencing, we provide evidence for the presence of rat lungworm (Angiostrongylus cantonensis) in Oklahoma, USA, and identified a potentially novel rat host (Sigmodon hispidus). Our results indicate a geographic range expansion for this medically and ecologically relevant parasite in North America.

ABSTRACT We analyzed the transcriptome of Escherichia coli K-12 by strand-specific RNA sequencing at single-nucleotide resolution during steady-state (logarithmic-phase) growth and upon entry into stationary phase in glucose minimal medium. To generate high-resolution transcriptome maps, we developed an organizational schema which showed that in practice only three features are required to define operon architecture: the promoter, terminator, and deep RNA sequence read coverage. We precisely annotated 2,122 promoters and 1,774 terminators, defining 1,510 operons with an average of 1.98 genes per operon. Our analyses revealed an unprecedented view of E. coli operon architecture. A large proportion (36%) of operons are complex with internal promoters or terminators that generate multiple transcription units. For 43% of operons, we observed differential expression of polycistronic genes, despite being in the same operons, indicating that E. coli operon architecture allows fine-tuning of gene expression. We found that 276 of 370 convergent operons terminate inefficiently, generating complementary 3′ transcript ends which overlap on average by 286 nucleotides, and 136 of 388 divergent operons have promoters arranged such that their 5′ ends overlap on average by 168 nucleotides. We found 89 antisense transcripts of 397-nucleotide average length, 7 unannotated transcripts within intergenic regions, and 18 sense transcripts that completely overlap operons on the opposite strand. Of 519 overlapping transcripts, 75% correspond to sequences that are highly conserved in E. coli (>50 genomes). Our data extend recent studies showing unexpected transcriptome complexity in several bacteria and suggest that antisense RNA regulation is widespread. IMPORTANCE We precisely mapped the 5′ and 3′ ends of RNA transcripts across the E. coli K-12 genome by using a single-nucleotide analytical approach. Our resulting high-resolution transcriptome maps show that ca. one-third of E. coli operons are complex, with internal promoters and terminators generating multiple transcription units and allowing differential gene expression within these operons. We discovered extensive antisense transcription that results from more than 500 operons, which fully overlap or extensively overlap adjacent divergent or convergent operons. The genomic regions corresponding to these antisense transcripts are highly conserved in E. coli (including Shigella species), although it remains to be proven whether or not they are functional. Our observations of features unearthed by single-nucleotide transcriptome mapping suggest that deeper layers of transcriptional regulation in bacteria are likely to be revealed in the future.