Chromosome flow sorted sequencing and assembly of chromosome 1H from barley accession Baronesse. Hordeum vulgare subsp. vulgare cultivar:Baronesse

Our goal is to resolve the Mla3 locus on chromosome 1H from the barley accession Baronesse. Chromosome flow sorting of Baronesse chromosome 1H was performed using the methods described by Dolezel et al. (2012) Functional and Integrative Genomics 12, 397-416 and chromosomal high molecular weight (HMW) DNA was prepared as described in Thind et al. (2017) Nature Biotechnology 35, 793-796. Chicago Dovetail sequencing of the chromosome was performed by Dovetail Genomics (Santa Cruz, CA, USA), with initial assembly in Meraculous and final scaffolding in HiRise. Briefly, Chicago libraries were generated by in vitro chromatin reconstitution using 250 ng HMW DNA. After fixation with formaldehyde, chromatin was digested with MboI, biotinylated nucleotides were added to 5 prime overhangs, and proximity ligation was performed. After reversal of crosslinking the DNA was treated to remove biotin and sheared to approximately 400 bp. A sequencing library was generated using NEBNext Ultra enzymes (New England Biolabs) and Illumina-compatible adapters and sequenced using Illumina HighSeq X using two insert sizes, 221 bp and 454 bp, with 266.3 and 246.9 million paired end reads, respectively. An initial assembly using Meraculous had length 450.7 Mb on 40,855 scaffolds. The HiRise assembly had 454.5 Mb on 2,009 scaffolds.

本研究旨在解析大麦品种Baronesse的1H染色体上的Mla3基因座（locus）。采用Dolezel等人（2012年，《功能与整合基因组学》（"Functional and Integrative Genomics"）第12卷，397-416页）报道的方法，对Baronesse的1H染色体进行流式分选；并参照Thind等人（2017年，《自然·生物技术》（"Nature Biotechnology"）第35卷，793-796页）的流程制备染色体高分子量（high molecular weight, HMW）DNA。该染色体的Chicago Dovetail测序（Chicago Dovetail sequencing）由美国加利福尼亚州圣克鲁斯的Dovetail Genomics公司完成，初始基因组组装采用Meraculous软件，最终支架搭建使用HiRise软件。简要而言，该测序文库通过体外染色体重构制备，使用250 ng的HMW DNA。经甲醛固定后，染色质经MboI酶切，向5'突出端添加生物素标记核苷酸，随后进行邻近连接反应。交联逆转后，对DNA进行去生物素处理，并剪切至约400 bp的片段。采用NEBNext Ultra酶（New England Biolabs）与Illumina兼容接头构建测序文库，使用Illumina HighSeq X测序平台完成测序，设置两种插入片段长度：221 bp与454 bp，分别获得266.3百万及246.9百万条双端读段（paired end reads）。采用Meraculous完成的初始组装共包含40855个支架，总长度达450.7 Mb。经HiRise完成的最终组装共包含2009个支架，总长度达454.5 Mb。