DOI: 10.5061/dryad.djh9w0w2g
title: Reference genome choice and filtering thresholds jointly influence phylogenomic analyses

description: Molecular phylogenies are a cornerstone of modern comparative biology and
 are commonly employed to investigate a range of biological phenomena, such
 as diversification rates, patterns in trait evolution, biogeography, and
 community assembly. Recent work has demonstrated that significant biases
 may be introduced into downstream phylogenetic analyses from processing
 genomic data; however, it remains unclear whether there are interactions
 among bioinformatic parameters or biases introduced through the choice of
 reference genome for sequence alignment and variant-calling. We address
 these knowledge gaps by employing a combination of simulated and empirical
 data sets to investigate to what extent the choice of reference genome in
 upstream bioinformatic processing of genomic data influences phylogenetic
 inference, as well as the way that reference genome choice interacts with
 bioinformatic filtering choices and phylogenetic inference method. We
 demonstrate that more stringent minor allele filters bias inferred trees
 away from the true species tree topology, and that these biased trees tend
 to be more imbalanced and have a higher center of gravity than the true
 trees. We find the greatest topological accuracy when filtering sites for
 minor allele count &gt; 3–4 in our 51-taxa data sets, while tree
 center of gravity was closest to the true value when filtering for sites
 with minor allele count &gt; 1-2. In contrast, filtering for missing
 data increased accuracy in the inferred topologies; however, this effect
 was small in comparison to the effect of minor allele filters and may be
 undesirable due to a subsequent mutation spectrum distortion. The bias
 introduced by these filters differs based on the reference genome used in
 short read alignment, providing further support that choosing a reference
 genome for alignment is an important bioinformatic decision with
 implications for downstream analyses. These results demonstrate that
 attributes of the study system and dataset (and their interaction) add
 important nuance for how best to assemble and filter short read genomic
 data for phylogenetic inference.
description: Data and supplementary material here are associated with the
 manuscript "Reference genome choice and filtering thresholds jointly
 influence phylogenetic analyses". Scripts can be found at https://github.com/jessicarick/refbias_scripts, and are archived on Zenodo at https://doi.org/10.5281/zenodo.5940690.
description: # Reference genome choice and filtering thresholds jointly influence
 phylogenomic analyses In addition to the supplemental methods, tables, and
 figures
 (**SUPPLEMENT\_Reference\_Bias\_in\_SNP\_based\_Phylogenetics\_SysBio.PDF**), there are four compressed directories included in this repository: 1. **simulation\_vcfs.tar.gz**: contains unfiltered VCFs for each of the simulation iterations, aligned to each of the reference genomes; each of these is named with the simulation date, tree height, simulation number, and reference genome identity (INT/EXT) 2. **empirical\_vcfs.tar.gz**: contains unfiltered VCFs for each of the empirical datasets (10 replicates for each clade), aligned to each of the reference genomes; each of these is named with the simulation number, and reference genome identity (INT/EXT) 3. **sim\_results\_raxml\_astral.tar.gz**: contains results for each combination of simulation, reference genome, and filtering scheme * "all-subsamp-raxml.trees" contains inferred RAxML phylogenies for the subsampled datasets; * "all-subsamp-raxml.names" contains the names for the inferred RAxML phylogenies for the subsampled datasets; * "all-raxml.trees" contains inferred RAxML phylogenies for the full datasets; * "all-raxml.names" contains names for the inferred RAxML phylogenies for the full datasets; * "all-astral.trees" contains inferred ASTRAL phylogenies for the full datasets; * "all-astral.names" contains names for inferred ASTRAL phylogenies for the full datasets; * "s\_tree.trees" contains the simulated species trees for each simulation; * "s\_tree.names" contains the names of the simulated species trees for each simulation; * "varSites" files contain the number of pre-filtering variable sites for each gene in each dataset; * "SNPs" files contain the number of post-filtering variable sites in each dataset * "TTR.gt\_rf" files contain the mean Robinson-Foulds distance between gene trees for the given simulation 4. **emp\_results\_raxml\_astral.tar.gz**: contains results for the empirical datasets * "cichlids-emp-batch.trees" contains inferred RAxML phylogenies for the Tropheine datasets; * "cichlids-emp-tree.names" contains the names for the inferred RAxML phylogenies for the Tropheine datasets; * "cichlids-emp-ASTRAL-batch.trees" contains inferred ASTRAL phylogenies for the Tropheine datasets; * "cichlids-emp-ASTRAL-tree.names" contains the names for the inferred ASTRAL phylogenies for the Tropheine datasets; * "lates-emp-batch.trees" contains inferred RAxML phylogenies for the *Lates* datasets; * "lates-emp-tree.names" contains the names for the inferred RAxML phylogenies for the *Lates* datasets; * "lates-emp-ASTRAL-batch.trees" contains inferred ASTRAL phylogenies for the *Lates* datasets; * "lates-emp-ASTRAL-tree.names" contains the names for the inferred ASTRAL phylogenies for the *Lates* datasets; * "SNPs-emp-lates" contains the number of post-filtering SNPs in each of the *Lates* datasets; * "cichlids-emp-SNPs" contains the number of post-filtering SNPs in each of the Tropheine datasets 5. **simphy\_trees.tar.gz**: contains all simulated species trees and gene trees for each simulation