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The impact of post-alignment processing procedures on whole-exome sequencing data
Murilo Guimarães Borges1,2,3, Helena Tadiello de Moraes1,2, Cristiane de Souza Rocha1,2
1Universidade Estadual de Campinas (UNICAMP), Faculdade de Ciências Médicas, Departamento de Genética Médica e Medicina Genômica, Campinas, SP, Brazil.
Genetics and Molecular Biology
|December 11, 2020
Summary
Post-alignment procedures improve DNA sequencing accuracy by minimizing false positives. Using reference variants from 1000 Genomes and LatinGen databases enhances variant calling in whole-exome sequencing data.
Area of Science:
- Genomics
- Bioinformatics
- Computational Biology
Background:
- Massive DNA sequencing generates large datasets where variant calling algorithms can misinterpret insertions and deletions, leading to false positives.
- Post-alignment procedures using known genetic variants as references can improve the accuracy of variant calling by realigning and recalibrating sequencing reads.
Purpose of the Study:
- To investigate the impact of using different sets of common variants as references for post-processing pipelines in whole-exome sequencing data.
- To evaluate the effectiveness of reference variant sets from the 1000 Genomes project and the Latin American Database of Genetic Variation (LatinGen) in minimizing false-positive variant calls.
Main Methods:
- Whole-exome sequencing data was analyzed using the Genome Analysis Toolkit.
- Post-processing procedures including local realignment and quality recalibration were performed using reference variants from 1000 Genomes and LatinGen.
- Variant calling was conducted with and without post-processing to compare the number and concordance of identified variants.
Main Results:
- A higher number of variants were identified when no post-processing was performed, suggesting a higher rate of false positives.
- Using reference variants from both 1000 Genomes and LatinGen resulted in a higher concordance rate compared to analyses without realignment or quality recalibration.
- The study identified that rare variants called without realignment or quality recalibration were likely false positives.
Conclusions:
- Post-alignment procedures, particularly local realignment and quality recalibration using established variant databases, are crucial for accurate variant calling in whole-exome sequencing.
- The use of common variants from resources like 1000 Genomes and LatinGen as references significantly improves the reliability of variant identification by reducing false positives.
- Implementing these post-processing steps leads to more parsimonious and accurate variant call sets, essential for downstream genetic analyses.
