Table 1.

Hyb-Seq target enrichment probe design and bioinformatics pipeline. A script combining and detailing the steps of the probe design process, Building_exon_probes.sh, is provided in the supplementary materials (Appendix S1 ^{(44.6KB, pdf)} ).

Steps	Description	Primary program or custom script
Probe design
Match	Find genome and transcriptome sequences with 99% identity.	BLATa
Filter	Retain single hits of substantial length.	Part of Building_exon_probes.shb
Cluster	Remove isoforms and loci sharing >90% identity.	CD-HIT-ESTc, grab_singleton_clusters.pyb
Filter	Retain loci with long exons summing to desired length.	blat_block_analyzer.pyb
Cluster	Remove exons sharing >90% identity.	CD-HIT-ESTc, grab_singleton_clusters.pyb
Short read processing and data analysis
Read processing	Adapter trimming, quality filtering	Trimmomaticd
Exon assembly	Reconstruct a sequence for each sample, for each exon.	YASRAe, Alignreadsf
Identify assembled contigs	If contig identity is unknown, identify which targeting exon(s) it corresponds to.	BLATa
Sequence alignment I: Collate exons	Cluster orthologous exons across samples.	assembled_exons_to_fasta.pyb
Sequence alignment II: Perform alignment	Align homologous bases within each exon.	MAFFTg
Concatenate exons	For each locus, concatenate the aligned exons.	catfasta2phyml.plh
Gene tree construction	For each locus, estimate the maximum likelihood gene tree.	RAxMLi
Species tree construction	Estimate the species tree from independent gene trees in a coalescent framework.	MP-ESTj

^aKent (2002).

^bNew scripts written for this protocol, an example data set, and any future updates are available at https://github.com/listonlab/.

^cLi and Godzik (2006).

^dBolger et al. (2014).

^eRatan (2009).

^fStraub et al. (2011).

^gKatoh and Toh (2008).

^hNylander (2011).

ⁱStamatakis (2006).

^jLiu et al. (2010).