Efficient and unique cobarcoding of second-generation sequencing reads from long DNA molecules enabling cost-effective and accurate sequencing, haplotyping, and de novo assembly.,Genome Research

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Efficient and unique cobarcoding of second-generation sequencing reads from long DNA molecules enabling cost-effective and accurate sequencing, haplotyping, and de novo assembly.
Genome Research ( IF 6.2 ) Pub Date : 2019-04-04 , DOI: 10.1101/gr.245126.118
Ou Wang _{1,

2,

3} , Robert Chin ₄ , Xiaofang Cheng _{1,

2} , Michelle Ka Yan Wu ₄ , Qing Mao ₄ , Jingbo Tang ₅ , Yuhui Sun _{1,

2} , Ellis Anderson ₄ , Han K Lam ₄ , Dan Chen _{1,

2} , Yujun Zhou _{1,

2} , Linying Wang _{1,

2} , Fei Fan _{1,

2} , Yan Zou _{1,

2} , Yinlong Xie ₅ , Rebecca Yu Zhang ₄ , Snezana Drmanac ₄ , Darlene Nguyen ₄ , Chongjun Xu _{1,

2,

4} , Christian Villarosa ₄ , Scott Gablenz ₄ , Nina Barua ₄ , Staci Nguyen ₄ , Wenlan Tian ₄ , Jia Sophie Liu ₄ , Jingwan Wang _{1,

2} , Xiao Liu _{1,

2} , Xiaojuan Qi _{1,

2} , Ao Chen _{1,

2} , He Wang _{1,

2} , Yuliang Dong _{1,

2} , Wenwei Zhang _{1,

2} , Andrei Alexeev ₄ , Huanming Yang _{1,

6} , Jian Wang _{1,

6} , Karsten Kristiansen _{1,

2,

3} , Xun Xu _{1,

2} , Radoje Drmanac _{1,

2,

4,

5} , Brock A Peters _{1,

2,

4,

5}

Affiliation

Here, we describe single-tube long fragment read (stLFR), a technology that enables sequencing of data from long DNA molecules using economical second-generation sequencing technology. It is based on adding the same barcode sequence to subfragments of the original long DNA molecule (DNA cobarcoding). To achieve this efficiently, stLFR uses the surface of microbeads to create millions of miniaturized barcoding reactions in a single tube. Using a combinatorial process, up to 3.6 billion unique barcode sequences were generated on beads, enabling practically nonredundant cobarcoding with 50 million barcodes per sample. Using stLFR, we demonstrate efficient unique cobarcoding of more than 8 million 20- to 300-kb genomic DNA fragments. Analysis of the human genome NA12878 with stLFR demonstrated high-quality variant calling and phase block lengths up to N50 34 Mb. We also demonstrate detection of complex structural variants and complete diploid de novo assembly of NA12878. These analyses were all performed using single stLFR libraries, and their construction did not significantly add to the time or cost of whole-genome sequencing (WGS) library preparation. stLFR represents an easily automatable solution that enables high-quality sequencing, phasing, SV detection, scaffolding, cost-effective diploid de novo genome assembly, and other long DNA sequencing applications.

中文翻译：

从长DNA分子读取第二代测序的高效独特的共条形码，可实现经济高效且准确的测序，单体型和从头组装。

在这里，我们介绍了单管长片段读取（stLFR）技术，该技术可使用经济的第二代测序技术对长DNA分子的数据进行测序。它基于在原始长DNA分子的亚片段中添加相同的条形码序列（DNA共条形码）。为了有效地实现此目的，stLFR使用微珠的表面在单个试管中产生数百万个微型条形码反应。使用组合过程，在珠子上最多生成了36亿条唯一的条形码序列，从而实现了几乎无冗余的共条形码，每个样品具有5000万个条形码。使用stLFR，我们证明了超过800万个20至300 kb基因组DNA片段的有效独特共条形码。使用stLFR对人类基因组NA12878的分析显示出高质量的变异检测和最大N50 34 Mb的相块长度。我们还演示了NA12878复杂结构变异的检测和完整的二倍体从头组装。这些分析都是使用单个stLFR文库进行的，其构建并未显着增加全基因组测序（WGS）文库制备的时间或成本。stLFR代表一种易于自动化的解决方案，可实现高质量测序，定相，SV检测，支架，经济高效的二倍体从头基因组组装以及其他长DNA测序应用。而且它们的构建并未显着增加全基因组测序（WGS）文库制备的时间或成本。stLFR代表一种易于自动化的解决方案，可实现高质量测序，定相，SV检测，支架，经济高效的二倍体从头基因组组装以及其他长DNA测序应用。而且它们的构建并未显着增加全基因组测序（WGS）文库制备的时间或成本。stLFR代表一种易于自动化的解决方案，可实现高质量测序，定相，SV检测，支架，经济高效的二倍体从头基因组组装以及其他长DNA测序应用。

更新日期：2019-11-01

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