DOI: 10.5061/dryad.298qm
title: Data from: A targeted next-generation sequencing toolkit for exon-based cichlid phylogenomics

description: Cichlid fishes (family Cichlidae) are models for evolutionary and
 ecological research. Massively parallel sequencing approaches have been
 successfully applied to study relatively recent diversification in groups
 of African and Neotropical cichlids, but such technologies have yet to be
 used for addressing larger scale phylogenetic questions of cichlid
 evolution. Here we describe a process for identifying putative single-copy
 exons from five African cichlid genomes and sequence the targeted exons
 for a range of divergent (&gt; tens of millions of years) taxa with
 probes designed from a single reference species (Oreochromis niloticus,
 Nile tilapia). Targeted sequencing of 923 exons across ten cichlid species
 that represent the family's major lineages and geographic
 distribution resulted in a complete taxon matrix of 564 exons (649,549
 bp), representing 559 genes. Maximum likelihood and Bayesian analyses in
 both species tree and concatenation frameworks yielded the same fully
 resolved and highly supported topology, which matched the expected
 backbone phylogeny of the major cichlid lineages. This work adds to the
 body of evidence that it is possible to use a relatively divergent
 reference genome for exon target design and successful capture across a
 broad phylogenetic range of species. Furthermore, our results show that
 the use of a third-party laboratory coupled with accessible bioinformatics
 tools make such phylogenomics projects feasible for research groups that
 lack direct access to genomics facilities. We expect that these resources
 will be used in further cichlid evolution studies and hope the protocols
 and identified targets will also be useful for phylogenetic studies of a
 wider range of organisms.
description: Andinoacara pulcher
 sorted BAM fileSorted BAM file for
 Andinoacara pulcher generated using SAMtools. Mapping and assembly of
 reads was conducted with bowtie2 version 2.1.0 using the 'very
 sensitive' preset parameters, with a maximum fragment length for
 paired-end alignments (-X) of 600, with the 923 tilapia exons as the
 reference.AndinoacaraStandaloneFiltered_Ref923_PairedEndandSingletonsAligned_Presetverysensitive.sorted.bamBiotoecus dicentrarchus sorted BAM fileSorted BAM file for Biotoecus dicentrarchus generated using SAMtools. Mapping and assembly of reads was conducted with bowtie2 version 2.1.0 using the 'very sensitive' preset parameters, with a maximum fragment length for paired-end alignments (-X) of 600, with the 923 tilapia exons as the reference.BiotoecusStandaloneFiltered_Ref923_PairedEndandSingletonsAligned_Presetverysensitive.sorted.bamCichla temensis sorted BAM fileSorted BAM file for Cichla temensis generated using SAMtools. Mapping and assembly of reads was conducted with bowtie2 version 2.1.0 using the 'very sensitive' preset parameters, with a maximum fragment length for paired-end alignments (-X) of 600, with the 923 tilapia exons as the reference.CichlaStandaloneFiltered_Ref923_PairedEndandSingletonsAligned_Presetverysensitive.sorted.bamCrenicichla frenata sorted BAM fileSorted BAM file for Crenicichla frenata generated using SAMtools. Mapping and assembly of reads was conducted with bowtie2 version 2.1.0 using the 'very sensitive' preset parameters, with a maximum fragment length for paired-end alignments (-X) of 600, with the 923 tilapia exons as the reference.CrenicichlaStandaloneFiltered_Ref923_PairedEndandSingletonsAligned_Presetverysensitive.sorted.bamEtroplus suratensis sorted BAM fileSorted BAM file for Etroplus suratensis generated using SAMtools. Mapping and assembly of reads was conducted with bowtie2 version 2.1.0 using the 'very sensitive' preset parameters, with a maximum fragment length for paired-end alignments (-X) of 600, with the 923 tilapia exons as the reference.EtroplusStandaloneFiltered_Ref923_PairedEndandSingletonsAligned_Presetverysensitive.sorted.bam‘Geophagus’ brasiliensis sorted BAM fileSorted BAM file for ‘Geophagus’ brasiliensis generated using SAMtools. Mapping and assembly of reads was conducted with bowtie2 version 2.1.0 using the 'very sensitive' preset parameters, with a maximum fragment length for paired-end alignments (-X) of 600, with the 923 tilapia exons as the reference.GeophagusStandaloneFiltered_Ref923_PairedEndandSingletonsAligned_Presetverysensitive.sorted.bamHerichthys cyanoguttatus sorted BAM fileSorted BAM file for Herichthys cyanoguttatus generated using SAMtools. Mapping and assembly of reads was conducted with bowtie2 version 2.1.0 using the 'very sensitive' preset parameters, with a maximum fragment length for paired-end alignments (-X) of 600, with the 923 tilapia exons as the reference.HerichthysStandaloneFiltered_Ref923_PairedEndandSingletonsAligned_Presetverysensitive.sorted.bamHeterochromis multidens sorted BAM fileSorted BAM file for Heterochromis multidens generated using SAMtools. Mapping and assembly of reads was conducted with bowtie2 version 2.1.0 using the 'very sensitive' preset parameters, with a maximum fragment length for paired-end alignments (-X) of 600, with the 923 tilapia exons as the reference.HeterochromisStandaloneFiltered_Ref923_PairedEndandSingletonsAligned_Presetverysensitive.sorted.bamReference sequences from Oreochromis niloticusList of 923 reference sequences (FASTA format) from Oreochromis niloticus used for mapping and assembly.Oreochromis_Refseq_923.fnaOreochromis niloticus sorted BAM fileSorted BAM file for Oreochromis niloticus generated using SAMtools. Mapping and assembly of reads was conducted with bowtie2 version 2.1.0 using the 'very sensitive' preset parameters, with a maximum fragment length for paired-end alignments (-X) of 600, with the 923 tilapia exons as the reference.OreochromisStandaloneFiltered_PairedEndandSingletonsAligned_Presetverysensitive.sorted.bamParatilapia polleni sorted BAM fileSorted BAM file for Paratilapia polleni generated using SAMtools. Mapping and assembly of reads was conducted with bowtie2 version 2.1.0 using the 'very sensitive' preset parameters, with a maximum fragment length for paired-end alignments (-X) of 600, with the 923 tilapia exons as the reference.ParatilapiaStandaloneFiltered_Ref923_PairedEndandSingletonsAligned_Presetverysensitive.sorted.bamAlignment files for exons captured for all ten speciesAlignment files for exons captured for all ten species plus the reference sequence from the Nile tilapia (Oreochromis niloticus). Each individual exon alignments was sorted into one of three categories: 'good', 'passable', or 'poor'. Only alignments in the categories 'good' and 'passable' categories were used for phylogenetic analysis. Included are the concatenated alignments used for Bayesian (MrBayes; nexus format) and maximum likelihood (RAxML; phylip format) phylogenetic analyses and individual exon alignments (phylip format) used in RAxML bootstrap analyses that were ultimately analyzed in a species tree framework using MP-EST.Ilves&Lopez-Fernandez.MER_alignments.zipSummary of stepsThis file describes the steps used to identify SCP exons across 5 cichlid genomes, and process Illumina MiSeq paired-end sequence data from 923 exons across a broad phylogenetic range of 10 cichlid species. This is most certainly not an elegant or efficient way to conduct these searches & analyses [efficiency could be greatly improved through more automation (i.e., use of scripts)], but the steps below are feasible by those with minimal programming expertise (and a lot of patience).Ilves&Lopez-Fernandez_SummaryOfSteps.txtSequence filesConsensus sequences in FASTA format for all exons captured, generated with the following quality filters: base and mapping qualities of at least 20 and a minimum depth of coverage of 10. Sequence positions that did not meet all of these criteria are coded as N. Only sequences with a minimum of 50 bases are included. Each file is named with the ENSEMBL exon ID for Oreochromis niloticus and the genus name of the taxon.Sequences_min50bases.zip