Chromosome conformation capture sequencing (Hi-C) is a powerful method to comprehensively interrogate the three-dimensional positioning of chromatin in the nucleus. The development of Hi-C can be traced back to successive increases in the resolution and throughput of chromosome conformation capture (3C) (Dekker et al., 2002). The basic workflow of 3C consists of (i) fixation of intact chromatin, usually by formaldehyde, (ii) cutting the fixed chromatin with a restriction enzyme, (iii) religation of sticky ends under diluted conditions to favor ligations between cross-linked fragments or those between random fragments and (iv) quantifying the number of ligations events between pairs of genomic loci (de Wit and de Laat, 2012). In the original 3C protocol, ligation frequency was measured by amplification of selected ligation junctions corresponding to a small number of genomic loci (‘one versus one’) through semi-quantitative PCR (Dekker et al., 2002). The chromosome conformation capture-on-chip (4C) and chromosome conformation capture carbon copy (5C) technologies then extended 3C to count ligation events in a ‘one versus many’ or ‘many versus many’ manner, respectively. Hi-C (Lieberman-Aiden et al., 2009) finally combined 3C with next-generation sequencing (Metzker, 2010). Here, before religation sticky ends are filled in with biotin-labeled nucleotide analogs to enrich for fragments with a ligation junction in a later step. The Hi-C libraries are then subjected to high-throughput sequencing and the resultant reads mapped to a reference genome, allowing the determination of contact probabilities in a ‘many versus many’ way with a resolution that is limited only by the distribution of restriction sites and the read depth. The first application of Hi-C was the elucidation of global chromatin folding principles in the human genome (Lieberman-Aiden et al., 2009). Similar efforts have since been carried out in other eukaryotic model species such as yeast (Duan et al., 2010), Drosophila (Sexton et al., 2012) and Arabidopsis (Grob et al., 2014; Wang et al., 2015; Liu et al., 2016). Other uses of Hi-C include the study of chromatin looping at high-resolution (Rao et al., 2014; Liu et al., 2016), of chromatin reorganization along the cell cycle (Naumova et al., 2013) and of differences in chromatin organization in mutant individuals (Feng et al., 2014). The tethered conformation capture protocol (TCC) (Kalhor et al., 2011) described here is a variant of the original Hi-C method (Lieberman-Aiden et al., 2009) and was adapted to Triticeae.

Recent advances in next-generation sequencing techniques allow the detection of a large number of SNPs and their use in a high throughput manner. However, Cleaved Amplified Polymorphic Sequences (CAPSs) still play a significant role as complement to other high throughput methods for SNP genotyping. Therefore, new methods focusing on the acceleration of this type of markers are highly desirable. The combination of the classical CAPS technique and a M13-tailed primer multiplexing assay was used to develop an agarose gel free protocol for the analysis of SNPs via restriction enzyme digestion. PCR products were fluorescence labeled with a universal M13 primer and subsequently digested with the appropriate restriction endonuclease. After mixing differently labeled products, they were detected on a capillary electrophoresis system. This method allows the cost-effective genotyping of several SNPs in a multiplexed manner at an overall low cost in a short period of time. Additionally, this method could be efficiently combined with the simultaneous detection of SSRs at the same electrophoresis run resulting in a procedure well suited for marker-based selection procedures, genotyping of mapping populations and the assay of genetic diversity.

Plant Sequence Capture is used for targeted resequencing of whole exomes (all exons of a genome) of complex genomes e.g. barley and its relatives (Mascher et al., 2013). Sequencing and computing costs are significantly reduced since only the greatly enriched and gene-coding part of the barley genome is targeted, that corresponds to only 1-2% of the entire genome. Thus, applications such as genetic diversity studies and the isolation of single genes (“cloning-by-sequencing”) are greatly facilitated. Here, a protocol is provided describing the construction of shotgun DNA libraries from genomic barley DNA for sequencing on the Illumina HiSeq/MiSeq systems. The shotgun DNA sequencing libraries are hybridized to an oligonucleotide pool (Exome Library) encompassing the whole exome of barley. The Exome Library is provided as a liquid array containing biotinylated probes (Roche/NimbleGen). Subsequently, genomic shotgun DNA fragments hybridized to the Exome Library are affinity-purified using streptavidin coated magnetic beads. The captured library is PCR-amplified and sequenced using high-throughput short read sequencing-by-synthesis.