Target capture sequencing for investigating repetitive DNA

With the advance of high-throughput sequencing, reduced-representation methods such as target capture sequencing (TCS) emerged as cost-efficient ways of gathering genomic information, particularly from coding regions. As the off-target reads from such sequencing are expected to be similar to genome skimming (GS), Costa et al. assessed the quality of repeat characterization in plant genomes using these data.

To check whether off-target reads could also be recycled for identifying and potentially quantifying repetitive DNA, the team selected the sedge Rhynchospora as a model. Rhynchospora is one of the largest genera of Cyperaceae, comprising ~350 species. Large-scale repeat analysis covering all major clades of Rhynchospora would provide valuable insights into the repeat evolution of this genus.

All the major repetitive DNA families were identified in TCS, including repeats that showed abundances as low as 0.01 % in the GS data. Rank correlations between GS and TCS repeat abundances were moderately high (r = 0.58–0.85), increasing after filtering out the targeted loci from the raw TCS reads (r = 0.66–0.92). Repeat data obtained by TCS were also reliable in developing a cytogenetic probe of a new variant of the holocentromeric satellite Tyba. Repeat-based phylogenies from TCS data were congruent with those obtained from GS data and the gene-alignment tree.

Costa et al. were able to find most of the repeat diversity of five Rhynchospora species using filtered and unfiltered TCS reads. Depending on the filtering strategy employed, the Rhynchospora data used here showed low abundance of on-target reads, indicating a considerable proportion of off-target reads suitable for repeat analysis.

In a commentary on the article, Heitkam and Garcia state: “[T]he repeats identified in the target capture datasets were effective for the development of fluorescent in situ hybridization (FISH) probes, indicating that the corresponding sequences were representative for the individual repeat families. This implies that repurposing of available target capture sequences can be an economic alternative for enabling large-scale cytogenomics, especially given the cost of genome skimming for a large number of species.”

“The identification of suitable cytogenetic markers for FISH, as proposed here, is especially promising and may enable larger, comparative cytogenetic studies at lower costs which could complement phylogenomics approaches. This combined cytogenomics and phylogenomics strategy will certainly generate new questions regarding the chromosomal contribution to speciation and diversification.”

RESEARCH ARTICLE

Costa, L., Marques, A., Buddenhagen, C., Thomas, W.W., Huettel, B., Schubert, V., Dodsworth, S., Houben, A., Souza, G., Pedrosa-Harand, A., 2021. Aiming off the target: recycling target capture sequencing reads for investigating repetitive DNA. Annals of Botany. https://doi.org/10.1093/aob/mcab063