Multifaceted Hi-C benchmarking: what makes a difference in chromosome-scale genome scaffolding?
Background: Hi-C, a derivative of chromosome conformation capture (3C) targeting the whole genome, was originally developed as a means for characterizing chromatin conformation. More recently, this method has also been frequently employed in elongating nucleotide sequences obtained by de novo genome sequencing and assembly, in which the number of resultant sequences rarely converge into the chromosome number. Despite the prevailing and irreplaceable use, sample preparation methods for Hi-C have not been intensively discussed, especially from the standpoint of genome scaffolding. Results: To gain insights into the best practice of Hi-C scaffolding, we performed a multifaceted methodological comparison using vertebrate samples and optimized various factors during sample preparation, sequencing, and computation. As a result, we have identified some key factors that help improve Hi-C scaffolding including the choice and preparation of tissues, library preparation conditions, and restriction enzyme(s), as well as the choice of scaffolding program and its usage. Conclusions: This study provides the first comparison of multiple sample preparation kits/protocols and computational programs for Hi-C scaffolding, by an academic third party. We introduce a customized protocol designated the 'inexpensive and controllable Hi-C (iconHi-C) protocol', in which the optimal conditions revealed by this study have been incorporated, and release the resultant chromosome-scale genome assembly of the Chinese softshell turtle Pelodiscus sinensis.
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