If the origins of fragments are known in genome sequencing projects, it is straightforward to reconstruct diploid consensus sequences. In reality, however, this is not true. Although there are proposed methods to reconstruct haplotypes from genome sequencing projects, an accuracy assessment is required to evaluate the confidence of the estimated diploid consensus sequences. In this paper, we define the confidence score of diploid consensus sequences. It requires the calculation of the likelihood of an assembly. To calculate the likelihood, we propose a linear time algorithm with respect to the number of polymorphic sites. The likelihood calculation and confidence score are used for further improvements of haplotype estimation in two directions. One direction is that low-scored phases are disconnected. The other direction is that, instead of using nominal frequency 1/2, the haplotype frequency is estimated to reflect the actual contribution of each haplotype. Our method was evaluated on the simulated data whose polymorphism rate (1.2 percent) was based on Ciona intestinalis. As a result, the high accuracy of our algorithm was indicated: The true positive rate of the haplotype estimation was greater than 97 percent.

A panel of 86 different Candida albicans isolates was subjected to multilocus sequence typing (MLST) in two laboratories to obtain sequence data for 10 published housekeeping gene fragments. Analysis of data for all possible combinations of five, six, seven, eight, and nine of the fragments showed that a set comprising the fragments AAT1a, ACC1, ADP1, MPIb, SYA1, VPS13, and ZWF1b was the smallest that yielded 86 unique diploid sequence types for the 86 isolates. This set is recommended for future MLST with C. albicans.

BACKGROUND: Lack of generalized guidelines for DNA flow cytometric analysis (FCM) may be the main reason for its limited use in the clinical management of breast cancer.
METHODS: After an initial interlaboratory reproducibility study (Round I), we concluded that it was the evaluation of the DNA histograms rather than the technical performance of the analysis that was the main reason for discordant results between laboratories. Guidelines for the interpretation of DNA histograms were therefore drawn up. We present here data from a new reproducibility study (Round II) using these guidelines.
RESULTS: For 10 laboratories also participating in Round I, use of the guidelines increased the concordance in DNA ploidy status from 89% to 100% for the 46 samples used in both rounds. The concordance rate for SPF also increased; mean r(s)-value increased from 0.81 to 0.88, and mean kappa value (lower two-thirds versus upper third versus not reported) increased from 0.55 to 0.71. Five new laboratories, participating only in Round II, also agreed with the 10 original laboratories regarding DNA ploidy status. With the inclusion of all 15 laboratories, we obtained a mean r(s)-value of 0.81 and a mean kappa value of 0.72 for SPF.
CONCLUSIONS: Generalized guidelines for DNA FCM increase interlaboratory agreement, which is highly important in clinical routines and in multicenter studies. Furthermore, inexperienced FCM laboratories using generalized guidelines can produce and interpret DNA FCM data equally as well as experienced laboratories.

From 1990-1996, 1,485 previously untreated invasive breast carcinomas were sampled by a pathologist for flow cytometric DNA analysis. The aim of the present work was to study the variations of flow cytometric DNA ploidy and S-phase evaluation according to the conditions of DNA histogram interpretation. Results obtained with the American Consensus guidelines of 1993 and the François Baclesse Department of Pathology\'s own guidelines are presented. According to the percentage of events taken into account to identify a DNA aneuploid peak, the proportion of DNA diploid cases can change from 35-39%. For S-phase evaluation, although the two guidelines were quite different, the results of S-phase cutoff were identical. Whichever guidelines were used, there was a strong relationship between DNA ploidy and/or S-phase and classical clinicopathological factors (T, N, histological type, grade, receptor status, or lymphatic invasion), with the exception of age, whose correlation was discrepant with S phase according to the set of guidelines. Whichever guidelines were used, ploidy and S phase correlated strongly with survival (overall, metastasis-free, or recurrence-free). Hence we recommend the use of the American consensus guidelines, despite minor imperfections, because they are now well-known, allow a high yield in the ratio of assessable S phases, and permit standardization in the technical processing and reporting of S phases, thanks to the use of terciles.

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