Next Generation Sequencing of Pooled Samples: Guideline for Variants' Filtering.
Sequencing large number of individuals, which is often needed for population genetics studies, is still economically challenging despite falling costs of Next Generation Sequencing (NGS). Pool-seq is an alternative cost- and time-effective option in which DNA from several individuals is pooled for sequencing. However, pooling of DNA creates new problems and challenges for accurate variant call and allele frequency (AF) estimation. In particular, sequencing errors confound with the alleles present at low frequency in the pools possibly giving rise to false positive variants. We sequenced 996 individuals in 83 pools (12 individuals/pool) in a targeted re-sequencing experiment. We show that Pool-seq AFs are robust and reliable by comparing them with public variant databases and in-house SNP-genotyping data of individual subjects of pools. Furthermore, we propose a simple filtering guideline for the removal of spurious variants based on the Kolmogorov-Smirnov statistical test. We experimentally validated our filters by comparing Pool-seq to individual sequencing data showing that the filters remove most of the false variants while retaining majority of true variants. The proposed guideline is fairly generic in nature and could be easily applied in other Pool-seq experiments.
Institute for Biomedical Technologies, National Research Council, Segrate (MI), Italy.|Department of Science and Technology, University of Sannio, Benevento, Italy.
Santosh
S
Anand
Institute for Biomedical Technologies, National Research Council, Segrate (MI), Italy.
Eleonora
E
Mangano
Interdisciplinary Research Center of Autoimmune Diseases IRCAD, University of Eastern Piedmont, Novara, Italy.|Department of Health Sciences, University of Eastern Piedmont, Novara, Italy.
Nadia
N
Barizzone
Institute for Biomedical Technologies, National Research Council, Segrate (MI), Italy.
Roberta
R
Bordoni
Laboratory of Human Genetics of Neurological Disorders, Institute of Experimental Neurology (INSPE), Division of Neuroscience, San Raffaele Scientific Institute, Milan, Italy.
Melissa
M
Sorosina
Laboratory of Human Genetics of Neurological Disorders, Institute of Experimental Neurology (INSPE), Division of Neuroscience, San Raffaele Scientific Institute, Milan, Italy.
Ferdinando
F
Clarelli
Interdisciplinary Research Center of Autoimmune Diseases IRCAD, University of Eastern Piedmont, Novara, Italy.|Department of Health Sciences, University of Eastern Piedmont, Novara, Italy.
Lucia
L
Corrado
Laboratory of Human Genetics of Neurological Disorders, Institute of Experimental Neurology (INSPE), Division of Neuroscience, San Raffaele Scientific Institute, Milan, Italy.|Department of Neurology, Division of Neuroscience, Scientific Institute San Raffaele, Milan, Italy.
Filippo
F
Martinelli Boneschi
Interdisciplinary Research Center of Autoimmune Diseases IRCAD, University of Eastern Piedmont, Novara, Italy.|Department of Health Sciences, University of Eastern Piedmont, Novara, Italy.
Sandra
S
D'Alfonso
Institute for Biomedical Technologies, National Research Council, Segrate (MI), Italy.
Gianluca
G
De Bellis
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