DOI: 10.5061/dryad.8030nq9
title: Data from: Genomic sequence capture of haemosporidian parasites: methods and prospects for enhanced study of host-parasite evolution

description: Avian malaria and related haemosporidians (Plasmodium, [Para]Haemoproteus,
 and Leucocytoozoon) represent an exciting multi-host, multi-parasite
 system in ecology and evolution. Global research in this field accelerated
 after 1) the publication in 2000 of PCR protocols to sequence a
 haemosporidian mitochondrial (mtDNA) barcode, and 2) the development in
 2009 of an open-access database to document the geographic and host ranges
 of parasite mtDNA haplotypes. Isolating haemosporidian nuclear DNA from
 bird hosts, however, has been technically challenging, slowing the
 transition to genomic-scale sequencing techniques. We extend a
 recently-developed sequence capture method to obtain hundreds of
 haemosporidian nuclear loci from wild bird samples, which typically have
 low levels of infection, or parasitemia. We tested 51 infected birds from
 Peru and New Mexico and evaluated locus recovery in light of variation in
 parasitemia, divergence from reference sequences, and pooling strategies.
 Our method was successful for samples with parasitemia as low as ~0.02% (2
 of 10,000 blood cells infected) and mtDNA divergence as high as 15.9% (one
 Leucocytozoon sample), and using the most cost-effective pooling strategy
 tested. Phylogenetic relationships estimated with &gt;300 nuclear loci
 were well resolved, providing substantial improvement over the mtDNA
 barcode. We provide protocols for sample preparation and sequence capture
 including custom probe sequences, and describe our bioinformatics pipeline
 using aTRAM 2.0, PHYLUCE, and custom Perl/Python scripts. This approach
 can be applied to thousands of avian samples that have already been found
 to have haemosporidian infections of at least moderate intensity, greatly
 improving our understanding of parasite speciation, biogeography, and
 evolutionary dynamics.
description: ScriptsScripts for processing avian haemosporidian sequence capture data. Example steps for the data processing pipeline are provided in the textfile 'example_pipeline_steps.txt', beginning from demultiplexed fastq files through RAxML alignments for phylogenetic analysis.Assemblies and Sequence DataAssemblies and sequence data for avian haemosporidians. The 'assemblies' directory contains the contigs from the last iteration of aTRAM 2.0 assemblies for all individuals processed. The 'stitched_exons' directory contains all sequences found for each gene using exon_stitching2.0; results are summarized by gene and by taxon in the 'Summary_stats...' CSV files. The 'individual_fastas' directory contains fasta sequences for the 15 successful individuals.Assemblies.zipAlignments and Phylogenetic AnalysesInput and output files from phylogenetic analyses using RAxML and SVDQuartets. Analyses with nuclear datasets were conducted with different levels of completeness (50%, 70%, 90% taxa present for each locus), and mitochondrial analyses were done separately. Analyses were also conducted with all successful individuals (n=19) or excluding the two individuals with mixed infections (n=17).Alignments_Trees.zip