Assessment of human diploid genome assembly with 10x Linked-Reads data

Abstract

14Background: Producing cost-effective haplotype-resolved personal genomes remains 15 challenging. 10x Linked-Read sequencing, with its high base quality and long-range information, 16 has been demonstrated to facilitate de novo assembly of human genomes and variant detection. 17In this study, we investigate in depth how the parameter space of 10x library preparation and 18 sequencing affects assembly quality, on the basis of both simulated and real libraries. 19Findings: We prepared and sequenced eight 10x libraries with a diverse set of parameters from 20 standard cell lines NA12878 and NA24385 and performed whole genome assembly on the data. 21We also developed the simulator LRTK-SIM to follow the workflow of 10x data generation and 22 produce realistic simulated Linked-Read data sets. We found that assembly quality could be 23 improved by increasing the total sequencing coverage (C) and keeping physical coverage of 24 DNA fragments (C F ) or read coverage per fragment (C R ) within broad ranges. The optimal 25 physical coverage was between 332X and 823X and assembly quality worsened if it increased 26 to greater than 1,000X for a given C. Long DNA fragments could significantly extend phase 27 blocks, but decreased contig contiguity. The optimal length-weighted fragment length (Wߤ ி ) 28 was around 50 -150kb. When broadly optimal parameters were used for library preparation 29 and sequencing, ca. 80% of the genome was assembled in a diploid state. 30Conclusion: The Linked-Read libraries we generated and the parameter space we identified 31 provide theoretical considerations and practical guidelines for personal genome assemblies 32 based on 10x Linked-Read sequencing. 33 Keywords: 10x Linked-Read sequencing, de novo assembly, diploid human genome, library 34 preparation 35 3 Data description 36 Introduction 37 The human genome holds the key for understanding the genetic basis of human evolution, 38 hereditary illnesses and many phenotypes. Whole-genome reconstruction and variant discovery, 39 accomplished by analysis of data from whole-genome sequencing experiments, are 40 foundational for the study of human genomic variation and analysis of genotype-phenotype 41 relationships. Over the past decades, cost-effective whole-genome sequencing has been 42 revolutionized by short-fragment approaches, the most widespread of which have been the 43 consistently improving generations of the original Solexa technology [1, 2], now referred to as 44 Illumina sequencing. Illumina's strengths and weaknesses are inherent in the sample 45 preparation and sequencing chemistry. Illumina generates short paired reads (2x150 base pairs 46 for the highest-throughput platforms) from short fragments (usually 400-500 base pairs) [3]. 47 Because many clonally amplified molecules generate a robust signal during the sequencing 48 reaction, Illumina's average per-base error rates are very low. 50The lack of long-range contiguity between end-sequenced short fragments limits their 51 application for reconstructing personal genomes. Long-rang...

A chromosome‐scale assembly of the black gram (Vigna mungo) genome

“…After sequencing, the barcodes are used to identify sequences that are in close proximity in the genome, and long DNA fragments can be reconstructed based on these linked reads (Ott et al., 2018). As the 10X Genomics linked‐read technology utilizes Illumina sequencing, it is more cost‐effective to generate the preliminary assembly using this approach compared to the long‐read PacBio sequencing (Zhang, Sun, et al, 2019; Zhang et al, 2019) . The preliminary assembly obtained from the 10X Genomics linked‐read strategy was 498.9 Mb with a scaffold N50 length of 5.2 Mb.…”

“…We also obtained Iso‐seq data from multiple tissues (leaf, root, stem, flower, 1‐week‐old pod and 3‐week‐old pod) and identified transcript variants exhibiting alternative splicing events. In contrast to the observations made in black gram, intron retention has been reported as the most prevalent alternative splicing mechanism in several plant species such as Arabidopsis (Marquez, Brown, Simpson, Barta, & Kalyna, 2012), G. max (Shen et al., 2014), V. radiata (Satyawan, Kim, & Lee, 2017), cotton (Feng, Xu, Liu, Cui, & Zhou, 2019), maize (Thatcher et al., 2016; Wang et al., 2016) and rice (Zhang, Sun, et al, 2019; Zhang, Zhou, et al, 2019). Our high‐quality genome assembly along with the genomic variation information from the germplasm provides an invaluable resource for investigating marker‐trait association at a whole genome level, gene expression analyses and comparative genomics and phylogenetic studies in legume species.…”

Aquila enables reference-assisted diploid personal genome assembly and comprehensive variant detection based on linked reads

“…To explore the performance of Aquila we used six libraries of 10X linked-read sequencing data that were previously generated by us 18 , 22 from gentle DNA preparations of NA12878 and NA24385 cell lines. The average inferred DNA fragment lengths and their distributions varied among libraries (Supplementary Table 1 ).…”

“…Due to the combination of high base pair-level sequence accuracy and long-range information, 10X/Illumina data therefore support excellent SNP and small indel detection and phasing 13 , as well as breakpoint detection of large events in cancer 13 , 15 , 16 . For diploid genome reconstruction, 10X developed the de novo assembler, Supernova, which has been shown to produce whole human genome assemblies from 56-fold coverage 10×/Illumina data 17 , 18 .…”

“…It then discovers the most important types of variation on the basis of pairwise alignment to the reference, and infers phasing for all types of assembled variants through previous long-range phasing information. We test its performance with six libraries of 10× linked-reads data for NA12878 and NA24385 individuals, which we had previously generated for evaluation of linked-read-based de novo assembly with Supernova 18 , 22 . We show that Aquila offers excellent small indel and SV detection at virtually no compromise for SNP detection, as well as highly accurate phasing of the vast majority of heterozygous variants, at reasonable reagent and computational costs.…”