Linked read technology for assembling large complex and polyploid genomes.

Short read DNA sequencing technologies have revolutionized genome assembly by providing high accuracy and throughput data at low cost. But it remains challenging to assemble short read data, particularly for large, complex and polyploid genomes. The linked read strategy has the potential to enhance the value of short reads for genome assembly because all reads originating from a single long molecule of DNA share a common barcode. However, the majority of studies to date that have employed linked reads were focused on human haplotype phasing and genome assembly. Here we describe a de novo maize B73 genome assembly generated via linked read technology which contains ~ 172,000 scaffolds with an N50 of 89 kb that cover 50% of the genome. Based on comparisons to the B73 reference genome, 91% of linked read contigs are accurately assembled. Because it was possible to identify errors with > 76% accuracy using machine learning, it may be possible to identify and potentially correct systematic errors. Complex polyploids represent one of the last grand challenges in genome assembly. Linked read technology was able to successfully resolve the two subgenomes of the recent allopolyploid, proso millet (Panicum miliaceum). Our assembly covers ~ 83% of the 1 Gb genome and consists of 30,819 scaffolds with an N50 of 912 kb. Our analysis provides a framework for future de novo genome assemblies using linked reads, and we suggest computational strategies that if implemented have the potential to further improve linked read assemblies, particularly for repetitive genomes.

Ott A ，Schnable JC ，Yeh CT ，Wu L ，Liu C ，Hu HC ，Dalgard CL ，Sarkar S ，Schnable PS ... -  《BMC GENOMICS》

A comprehensive investigation of metagenome assembly by linked-read sequencing.

The human microbiota are complex systems with important roles in our physiological activities and diseases. Sequencing the microbial genomes in the microbiota can help in our interpretation of their activities. The vast majority of the microbes in the microbiota cannot be isolated for individual sequencing. Current metagenomics practices use short-read sequencing to simultaneously sequence a mixture of microbial genomes. However, these results are in ambiguity during genome assembly, leading to unsatisfactory microbial genome completeness and contig continuity. Linked-read sequencing is able to remove some of these ambiguities by attaching the same barcode to the reads from a long DNA fragment (10-100 kb), thus improving metagenome assembly. However, it is not clear how the choices for several parameters in the use of linked-read sequencing affect the assembly quality. We first examined the effects of read depth (C) on metagenome assembly from linked-reads in simulated data and a mock community. The results showed that C positively correlated with the length of assembled sequences but had little effect on their qualities. The latter observation was corroborated by tests using real data from the human gut microbiome, where C demonstrated minor impact on the sequence quality as well as on the proportion of bins annotated as draft genomes. On the other hand, metagenome assembly quality was susceptible to read depth per fragment (C) and DNA fragment physical depth (C). For the same C, deeper C resulted in more draft genomes while deeper C improved the quality of the draft genomes. We also found that average fragment length (μ) had marginal effect on assemblies, while fragments per partition (N) impacted the off-target reads involved in local assembly, namely, lower N values would lead to better assemblies by reducing the ambiguities of the off-target reads. In general, the use of linked-reads improved the assembly for contig N50 when compared to Illumina short-reads, but not when compared to PacBio CCS (circular consensus sequencing) long-reads. We investigated the influence of linked-read sequencing parameters on metagenome assembly comprehensively. While the quality of genome assembly from linked-reads cannot rival that from PacBio CCS long-reads, the case for using linked-read sequencing remains persuasive due to its low cost and high base-quality. Our study revealed that the probable best practice in using linked-reads for metagenome assembly was to merge the linked-reads from multiple libraries, where each had sufficient C but a smaller amount of input DNA. Video Abstract.

Zhang L ，Fang X ，Liao H ，Zhang Z ，Zhou X ，Han L ，Chen Y ，Qiu Q ，Li SC ... -  《Microbiome》

Highly accurate long reads are crucial for realizing the potential of biodiversity genomics.

Generating the most contiguous, accurate genome assemblies given available sequencing technologies is a long-standing challenge in genome science. With the rise of long-read sequencing, assembly challenges have shifted from merely increasing contiguity to correctly assembling complex, repetitive regions of interest, ideally in a phased manner. At present, researchers largely choose between two types of long read data: longer, but less accurate sequences, or highly accurate, but shorter reads (i.e., >Q20 or 99% accurate). To better understand how these types of long-read data as well as scale of data (i.e., mean length and sequencing depth) influence genome assembly outcomes, we compared genome assemblies for a caddisfly, Hesperophylax magnus, generated with longer, but less accurate, Oxford Nanopore (ONT) R9.4.1 and highly accurate PacBio HiFi (HiFi) data. Next, we expanded this comparison to consider the influence of highly accurate long-read sequence data on genome assemblies across 6750 plant and animal genomes. For this broader comparison, we used HiFi data as a surrogate for highly accurate long-reads broadly as we could identify when they were used from GenBank metadata. HiFi reads outperformed ONT reads in all assembly metrics tested for the caddisfly data set and allowed for accurate assembly of the repetitive ~ 20 Kb H-fibroin gene. Across plants and animals, genome assemblies that incorporated HiFi reads were also more contiguous. For plants, the average HiFi assembly was 501% more contiguous (mean contig N50 = 20.5 Mb) than those generated with any other long-read data (mean contig N50 = 4.1 Mb). For animals, HiFi assemblies were 226% more contiguous (mean contig N50 = 20.9 Mb) versus other long-read assemblies (mean contig N50 = 9.3 Mb). In plants, we also found limited evidence that HiFi may offer a unique solution for overcoming genomic complexity that scales with assembly size. Highly accurate long-reads generated with HiFi or analogous technologies represent a key tool for maximizing genome assembly quality for a wide swath of plants and animals. This finding is particularly important when resources only allow for one type of sequencing data to be generated. Ultimately, to realize the promise of biodiversity genomics, we call for greater uptake of highly accurate long-reads in future studies.

Hotaling S ，Wilcox ER ，Heckenhauer J ，Stewart RJ ，Frandsen PB ... -  《BMC GENOMICS》

Genome assembly using Nanopore-guided long and error-free DNA reads.

Long-read sequencing technologies were launched a few years ago, and in contrast with short-read sequencing technologies, they offered a promise of solving assembly problems for large and complex genomes. Moreover by providing long-range information, it could also solve haplotype phasing. However, existing long-read technologies still have several limitations that complicate their use for most research laboratories, as well as in large and/or complex genome projects. In 2014, Oxford Nanopore released the MinION® device, a small and low-cost single-molecule nanopore sequencer, which offers the possibility of sequencing long DNA fragments. The assembly of long reads generated using the Oxford Nanopore MinION® instrument is challenging as existing assemblers were not implemented to deal with long reads exhibiting close to 30% of errors. Here, we presented a hybrid approach developed to take advantage of data generated using MinION® device. We sequenced a well-known bacterium, Acinetobacter baylyi ADP1 and applied our method to obtain a highly contiguous (one single contig) and accurate genome assembly even in repetitive regions, in contrast to an Illumina-only assembly. Our hybrid strategy was able to generate NaS (Nanopore Synthetic-long) reads up to 60 kb that aligned entirely and with no error to the reference genome and that spanned highly conserved repetitive regions. The average accuracy of NaS reads reached 99.99% without losing the initial size of the input MinION® reads. We described NaS tool, a hybrid approach allowing the sequencing of microbial genomes using the MinION® device. Our method, based ideally on 20x and 50x of NaS and Illumina reads respectively, provides an efficient and cost-effective way of sequencing microbial or small eukaryotic genomes in a very short time even in small facilities. Moreover, we demonstrated that although the Oxford Nanopore technology is a relatively new sequencing technology, currently with a high error rate, it is already useful in the generation of high-quality genome assemblies.

Madoui MA ，Engelen S ，Cruaud C ，Belser C ，Bertrand L ，Alberti A ，Lemainque A ，Wincker P ，Aury JM ... -  《BMC GENOMICS》

Assembly of chloroplast genomes with long- and short-read data: a comparison of approaches using Eucalyptus pauciflora as a test case.

Chloroplasts are organelles that conduct photosynthesis in plant and algal cells. The information chloroplast genome contained is widely used in agriculture and studies of evolution and ecology. Correctly assembling chloroplast genomes can be challenging because the chloroplast genome contains a pair of long inverted repeats (10-30 kb). Typically, it is simply assumed that the gross structure of the chloroplast genome matches the most commonly observed structure of two single-copy regions separated by a pair of inverted repeats. The advent of long-read sequencing technologies should remove the need to make this assumption by providing sufficient information to completely span the inverted repeat regions. Yet, long-reads tend to have higher error rates than short-reads, and relatively little is known about the best way to combine long- and short-reads to obtain the most accurate chloroplast genome assemblies. Using Eucalyptus pauciflora, the snow gum, as a test case, we evaluated the effect of multiple parameters, such as different coverage of long-(Oxford nanopore) and short-(Illumina) reads, different long-read lengths, different assembly pipelines, with a view to determining the most accurate and efficient approach to chloroplast genome assembly. Hybrid assemblies combining at least 20x coverage of both long-reads and short-reads generated a single contig spanning the entire chloroplast genome with few or no detectable errors. Short-read-only assemblies generated three contigs (the long single copy, short single copy and inverted repeat regions) of the chloroplast genome. These contigs contained few single-base errors but tended to exclude several bases at the beginning or end of each contig. Long-read-only assemblies tended to create multiple contigs with a much higher single-base error rate. The chloroplast genome of Eucalyptus pauciflora is 159,942 bp, contains 131 genes of known function. Our results suggest that very accurate assemblies of chloroplast genomes can be achieved using a combination of at least 20x coverage of long- and short-reads respectively, provided that the long-reads contain at least ~5x coverage of reads longer than the inverted repeat region. We show that further increases in coverage give little or no improvement in accuracy, and that hybrid assemblies are more accurate than long-read-only or short-read-only assemblies.

Wang W ，Schalamun M ，Morales-Suarez A ，Kainer D ，Schwessinger B ，Lanfear R ... -  《BMC GENOMICS》