Evaluating the impact of scoring parameters on the structure of intra-specific genetic variation using RawGeno, an R package for automating AFLP scoring

<p>Abstract</p> <p>Background</p> <p>Since the transfer and application of modern sequencing technologies to the analysis of amplified fragment-length polymorphisms (AFLP), evolutionary biologists have included an increasing number of samples and markers in their studies. Although justified in this context, the use of automated scoring procedures may result in technical biases that weaken the power and reliability of further analyses.</p> <p>Results</p> <p>Using a new scoring algorithm, RawGeno, we show that scoring errors – in particular "bin oversplitting" (i.e. when variant sizes of the same AFLP marker are not considered as homologous) and "technical homoplasy" (i.e. when two AFLP markers that differ slightly in size are mistakenly considered as being homologous) – induce a loss of discriminatory power, decrease the robustness of results and, in extreme cases, introduce erroneous information in genetic structure analyses. In the present study, we evaluate several descriptive statistics that can be used to optimize the scoring of the AFLP analysis, and we describe a new statistic, the information content per bin (I_bin) that represents a valuable estimator during the optimization process. This statistic can be computed at any stage of the AFLP analysis without requiring the inclusion of replicated samples. Finally, we show that downstream analyses are not equally sensitive to scoring errors. Indeed, although a reasonable amount of flexibility is allowed during the optimization of the scoring procedure without causing considerable changes in the detection of genetic structure patterns, notable discrepancies are observed when estimating genetic diversities from differently scored datasets.</p> <p>Conclusion</p> <p>Our algorithm appears to perform as well as a commercial program in automating AFLP scoring, at least in the context of population genetics or phylogeographic studies. To our knowledge, RawGeno is the only freely available public-domain software for fully automated AFLP scoring, from electropherogram files to user-defined working binary matrices. RawGeno was implemented in an R CRAN package (with an user-friendly GUI) and can be found at <url>http://sourceforge.net/projects/rawgeno</url>.</p

Improved Sensitivity in Comparative Genomic Hybridization Analysis of DNA Heteroploid Cell Mixtures after Pre-Enrichment of Subpopulations by Fluorescence Activated Cell Sorting

Cytogenetic analysis of solid tumors with comparative genomic hybridization (CGH) is hampered by the dilution of DNA from individual tumor subpopulations with DNA from other cells. We investigated to what extent this dilution effect can be alleviated using fluorescence activated cell sorting (flow sorting) of experimental DNA heteroploid cell mixtures prior to CGH. From mixtures of normal lymphocytes with triploid K‐562 cells the individual components were sorted according to stemline DNA content and processed by CGH in comparison with pure K‐562 samples and the original mixtures. Compared with 30 autosome copy number imbalances found in pure K‐562 samples, a mixture with 32% K‐562 cells showed 16 imbalancies, and none were detected in mixtures with 13% or 5% K‐562 cells. In contrast, 29, 22 and 23 imbalances were detected in K‐562 nuclei sorted from the 32%, 13% and 5% mixtures, respectively. This indicate that CGH analysis of flow sorted DNA aneuploid subpopulations enables a specific cytogenetic analysis of the individual subclones in a DNA heteroploid cell population

Improved sensitivity in comparative genomic hybridization analysis of DNA heteroploid cell mixtures after pre-enrichment of subpopulations by fluorescence activated cell sorting

Cytogenetic analysis of solid tumors with comparative genomic hybridization (CGH) is hampered by the dilution of DNA from individual tumor subpopulations with DNA from other cells. We investigated to what extent this dilution effect can be alleviated using fluorescence activated cell sorting (flow sorting) of experimental DNA heteroploid cell mixtures prior to CGH. From mixtures of normal lymphocytes with triploid K-562 cells the individual components were sorted according to stemline DNA content and processed by CGH in comparison with pure K-562 samples and the original mixtures. Compared with 30 autosome copy number imbalances found in pure K-562 samples, a mixture with 32% K-562 cells showed 16 imbalancies, and none were detected in mixtures with 13% or 5% K-562 cells. In contrast, 29, 22 and 23 imbalances were detected in K-562 nuclei sorted from the 32%, 13% and 5% mixtures, respectively. This indicate * Correspondence to: Jacob Larsen, Finsen Laboratory, Finsen Center, Rigshospitalet, Dept. 8621, Strandboulevarden 49, DK-2100 Copenhagen, Denmark. Tel.: +45 3545 5751; Fax: +45 3538 5450; E-mail: [email protected]. that CGH analysis of flow sorted DNA aneuploid subpopulations enables a specific cytogenetic analysis of the individual subclones in a DNA heteroploid cell population