Biodiversity, phylogeography and population genetic studies will be revolutionized by access to large datasets thanks to next generation sequencing methods. In this study, we develop an easy and cost-effective protocol for in-solution enrichment hybridization capture of complete chloroplast genomes applicable at deep multiplexed levels. The protocol uses cheap in-house species-specific probes developed via long range PCR of the entire chloroplast. Barcoded libraries are constructed and in-solution enrichment of the chloroplasts is done using the probes. This protocol was tested and validated on six economically important West African crop species, namely African rice, pearl millet, three African yam species and fonio. For pearl millet, we also demonstrate the effectiveness of this protocol to retrieve 95% of the sequence of the whole chloroplast on 95 multiplexed individuals in a single MiSeq run at a success rate of 95%. This new protocol allows whole chloroplast genomes to be retrieved at a modest cost and will allow unprecedented resolution for closely related species in phylogeography studies using plastomes.
replace95bam_and_ref
95bam_and_ref.zip
De novo assembly Chloroplast of Dioscorea rotundata
The software MITObim (v. 1.5, Hahn et al. 2013) was used to reconstruct de novo plastid genomes. We used as initial references: Dioscorea elephantipes (EF380353.1)
cpRotundatav1_28itcp.fasta
De novo assembly Chloroplast of Digitaria excilis
The software MITObim (v. 1.5, Hahn et al. 2013) was used to reconstruct de novo plastid genomes. We used as initial references: Pennisetum glaucum
cpFONIO-18itv1.fasta
De novo assembly Chloroplast of Cenchrus americanus
The software MITObim (v. 1.5, Hahn et al. 2013) was used to reconstruct de novo plastid genomes. We used as initial references: Zea mays
cpmilv3100it.fasta
RAM63_forward_500k
This file contain 500k forward reads Illumina TrueSeq v3 75 PE, from RAM63, an O. sativa ssp indica.These data have been used to validate the assembly method.