Analysis of sequence-tagged-connector strategies for DNA sequencing

The BAC-end sequencing, or sequence-tagged-connector (STC), approach to genome sequencing involves sequencing the ends of BAC inserts to scatter sequence tags (STCs) randomly across the genome. Once any BAC or other large segment of DNA is sequenced to completion by conventional shotgun approaches, these STC tags can be used to identify a minimum tiling path of BAC clones overlapping the nucleation sequence for sequence extension. Here, we explore the properties of STC-sequencing strategies within a mathematical model of a random target with homologous repeats and imperfect sequencing technology to understand the consequences of varying various parameters on the incidence of problem clones and the cost of the sequencing project. Problem clones are defined as clones for which either (A) there is no identifiable overlapping STC to extend the sequence in a particular direction or (B) the identified STC with minimum overlap comes from a nonoverlapping clone, either owing to random false matches or repeat-family homology. Based on the minimum overlap, we estimate the number of clones to be entirely sequenced and, then, using cost estimates, identify the decision rule (the degree of sequence similarity required before a match is declared between an STC and a clone) to minimize overall sequencing cost. A method to optimize the overlap decision rule is highly desirable, because both the total cost and the number of problem clones are shown to be highly sensitive to this choice. For a target of 3 Gb containing approximately 800 Mb of repeats with 85%-90% identity, we expect <10 problem clones with 15 times coverage by 150-kb clones. We derive the optimal redundancy and insert sizes of clone libraries for sequencing genomes of various sizes, from microbial to human. We estimate that establishing the resource of STCs as a means of identifying minimally overlapping clones represents only 1%-3% of the total cost of sequencing the human genome, and, up to a point of diminishing returns, a larger STC resource is associated with a smaller total sequencing cost.     

Lowering the cut off value of an automated chlamydia enzyme immunoassay and confirmation by PCR and direct immunofluorescent antibody test.

AIMS: To increase the sensitivity of an automated chlamydia enzyme immunoassay by significantly lowering its cut off value, and to maintain specificity by confirmation with polymerase chain reaction (PCR) and direct immunofluorescent antibody test (DFA). METHODS: Over five months, the cut off value of the enzyme immunoassay used to screen urogenital samples for chlamydia antigen was reduced from 80 to 10. Samples with a test value of 10 or above were further tested with a commercial PCR assay. All samples during the first three months and discrepant samples during the last two months of the study were also tested with the DFA. RESULTS: 3250 urogenital swabs (1246 urethral, 1335 endocervical, 669 pooled urethral/endocervical) from 1246 males and 2004 females were processed. Using the manufacturer's recommended cut off of 80, the enzyme immunoassay identified chlamydia antigen in 134 samples (4.1%). Using the lower cut off value of 10 and either PCR or DFA as the confirmatory test, Chlamydia trachomatis was identified in 178 samples (5.5%). Thus, 45 additional positive samples were identified and the confirmed detection rate was increased by 33.8% (45/133). Excluding equivocal PCR results, the concordance between DFA and PCR was 91.8%. This strategy increased the detection rate by 2.1% in men and 0.9% in women (significant only in men). In female patients, pooled urethral/endocervical swabs as a specimen gave a significantly higher yield than endocervical swabs regardless of whether the lower cut off strategy was used. CONCLUSIONS: This strategy of significantly lowering the cut off test value with confirmation on the same specimen by either PCR or DFA is feasible and cost effective. The use of pooled urethral/ endocervical specimens in females should be considered routinely as detection rate was significantly improved.     

Chemical and biological weapons: new questions, new answers

The words "chemical and biological weapons" (CBW) send a shiver down most spines these days. With the end of the Cold War, the possibility of a massive nuclear confrontation appears remote, so today many popular doomsday scenarios center on the aggressive use of chemical or biological warfare by rogue nations or terrorist groups. As exaggerated as some of the accounts are, with CBW cast as the latest unseen, unstoppable enemy, the threat posed by these weapons is all too real, and growing.     

Antibiotic resistance in nosocomial isolates in Scotland

In this first multi-centre study in Scotland, 1028 consecutive Gram-negative and staphylococci strains were obtained from four major teaching hospitals. E. coli was the most common organism among both intensive care units (ICUs) (39%) and non-ICU strains (46.6%). The prevalence of antibiotic resistance among E. coli was always higher in isolates from ICUs than non-ICUs: ceftazidime (14.1%, 7.2%), ceftriaxone (12.7%, 6.1%), cefotaxime (15.5%, 8.7%), cefuroxime (28.8%, 20.8%), amoxicillin plus clavulanic acid (52.2%, 38.8%) and gentamicin (7.0%, 2.8%). The highest incidences of resistance were identified among Enterobacter/Citrobacter spp. from ICUs; 43.8%, 41.7%, 45.8%, 54.2%, 87.5% and 10.4% of these organisms were resistant to ceftazidime, ceftriaxone, cefotaxime, cefuroxime, amoxicillin plus clavulanic acid and gentamicin, respectively.     

Comparison of Electromyographic Activity in Normal Lumbar Sacrospinalis Musculature during continuous and Intermittent Pelvic Traction

This study investigates whether there is a difference in electromyographic activity in the lumbar sacrospinalis musculature during continuous and intermittent pelvic traction. Twenty-nine normal subjects were randomly assigned to a control group, a continuous traction group, or an intermittent traction group. Electromyographic activity was recorded at specific timed intervals. Myoelectric activity increased with the onset of either type of traction, but by the third recording both groups had returned to their normal initial resting myoelectric recordings. The myoelectric patterns over time were similar for the two treatment groups. No significant difference in electromyographic activity of the lumbar sacrospinalis musculature during intermittent or continuous pelvic traction was found. J Orthop Sports Phys Ther 1981;2(3):137-141.     

Situational factors associated with AIDS risk behavior lapses and coping strategies used by gay men who successfully avoid lapses

While most gay men have reduced behavior practices at high risk for HIV infection, there is growing evidence that many also lapse to unsafe sex. This study examined situational factors related to risk behavior lapses as well as coping strategies used by men who successfully resist lapse urges. A convenience sample of 470 men patronizing gay bars or attending social organization meetings in four cities was surveyed. Forty-five percent of men were classified as "lapsers" (those who had had unprotected anal intercourse in the previous 6 months) and 24% were classified as "resisters" (those who successfully resisted urges to engage in this behavior). All provided information concerning the importance of factors related to the most recent occurrence of either unsafe sex or resisting unsafe urges. Most episodes of unsafe sex occurred outside monogamous relationships and with partners of unknown HIV serostatus, although simply inquiring about partner serostatus was relatively common. Lapsers rated affectionate feelings and wishing to please a partner as well as spontaneity of unsafe sex as the most important situational factors surrounding high-risk behavior. Resisters of unsafe sex urges reported active cognitive self-guidance, experience in safe sex, and recall of both AIDS fears and safety benefits as their most important coping strategies. Gay men who continue high-risk behavior may be overrelying on partner reports of negative serostatus. Lapse prevention approaches tailored to situations that create increased risk vulnerability must be developed. Teaching skills already used by men who successfully resist unsafe sex urges might be one approach.