August 19, 2021  |  

Case Study: Pioneering a pan-genome reference collection

At DuPont Pioneer, DNA sequencing is paramount for R&D to reveal the genetic basis for traits of interest in commercial crops such as maize, soybean, sorghum, sunflower, alfalfa, canola, wheat, rice, and others. They cannot afford to wait the years it has historically taken for high-quality reference genomes to be produced. Nor can they rely on a single reference to represent the genetic diversity in its germplasm.

April 21, 2020  |  

Tandem repeats lead to sequence assembly errors and impose multi-level challenges for genome and protein databases.

The widespread occurrence of repetitive stretches of DNA in genomes of organisms across the tree of life imposes fundamental challenges for sequencing, genome assembly, and automated annotation of genes and proteins. This multi-level problem can lead to errors in genome and protein databases that are often not recognized or acknowledged. As a consequence, end users working with sequences with repetitive regions are faced with ‘ready-to-use’ deposited data whose trustworthiness is difficult to determine, let alone to quantify. Here, we provide a review of the problems associated with tandem repeat sequences that originate from different stages during the sequencing-assembly-annotation-deposition workflow, and that may proliferate in public database repositories affecting all downstream analyses. As a case study, we provide examples of the Atlantic cod genome, whose sequencing and assembly were hindered by a particularly high prevalence of tandem repeats. We complement this case study with examples from other species, where mis-annotations and sequencing errors have propagated into protein databases. With this review, we aim to raise the awareness level within the community of database users, and alert scientists working in the underlying workflow of database creation that the data they omit or improperly assemble may well contain important biological information valuable to others. © The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.

September 22, 2019  |  

PacBio sequencing and its applications.

Single-molecule, real-time sequencing developed by Pacific BioSciences offers longer read lengths than the second-generation sequencing (SGS) technologies, making it well-suited for unsolved problems in genome, transcriptome, and epigenetics research. The highly-contiguous de novo assemblies using PacBio sequencing can close gaps in current reference assemblies and characterize structural variation (SV) in personal genomes. With longer reads, we can sequence through extended repetitive regions and detect mutations, many of which are associated with diseases. Moreover, PacBio transcriptome sequencing is advantageous for the identification of gene isoforms and facilitates reliable discoveries of novel genes and novel isoforms of annotated genes, due to its ability to sequence full-length transcripts or fragments with significant lengths. Additionally, PacBio’s sequencing technique provides information that is useful for the direct detection of base modifications, such as methylation. In addition to using PacBio sequencing alone, many hybrid sequencing strategies have been developed to make use of more accurate short reads in conjunction with PacBio long reads. In general, hybrid sequencing strategies are more affordable and scalable especially for small-size laboratories than using PacBio Sequencing alone. The advent of PacBio sequencing has made available much information that could not be obtained via SGS alone. Copyright © 2015 The Authors. Production and hosting by Elsevier Ltd.. All rights reserved.

September 22, 2019  |  

Genomic architecture of haddock (Melanogrammus aeglefinus) shows expansions of innate immune genes and short tandem repeats.

Increased availability of genome assemblies for non-model organisms has resulted in invaluable biological and genomic insight into numerous vertebrates, including teleosts. Sequencing of the Atlantic cod (Gadus morhua) genome and the genomes of many of its relatives (Gadiformes) demonstrated a shared loss of the major histocompatibility complex (MHC) II genes 100 million years ago. An improved version of the Atlantic cod genome assembly shows an extreme density of tandem repeats compared to other vertebrate genome assemblies. Highly contiguous assemblies are therefore needed to further investigate the unusual immune system of the Gadiformes, and whether the high density of tandem repeats found in Atlantic cod is a shared trait in this group.Here, we have sequenced and assembled the genome of haddock (Melanogrammus aeglefinus) – a relative of Atlantic cod – using a combination of PacBio and Illumina reads. Comparative analyses reveal that the haddock genome contains an even higher density of tandem repeats outside and within protein coding sequences than Atlantic cod. Further, both species show an elevated number of tandem repeats in genes mainly involved in signal transduction compared to other teleosts. A characterization of the immune gene repertoire demonstrates a substantial expansion of MCHI in Atlantic cod compared to haddock. In contrast, the Toll-like receptors show a similar pattern of gene losses and expansions. For the NOD-like receptors (NLRs), another gene family associated with the innate immune system, we find a large expansion common to all teleosts, with possible lineage-specific expansions in zebrafish, stickleback and the codfishes.The generation of a highly contiguous genome assembly of haddock revealed that the high density of short tandem repeats as well as expanded immune gene families is not unique to Atlantic cod – but possibly a feature common to all, or most, codfishes. A shared expansion of NLR genes in teleosts suggests that the NLRs have a more substantial role in the innate immunity of teleosts than other vertebrates. Moreover, we find that high copy number genes combined with variable genome assembly qualities may impede complete characterization of these genes, i.e. the number of NLRs in different teleost species might be underestimates.

July 19, 2019  |  

Rapid detection of expanded short tandem repeats in personal genomics using hybrid sequencing.

Long expansions of short tandem repeats (STRs), i.e. DNA repeats of 2-6 nt, are associated with some genetic diseases. Cost-efficient high-throughput sequencing can quickly produce billions of short reads that would be useful for uncovering disease-associated STRs. However, enumerating STRs in short reads remains largely unexplored because of the difficulty in elucidating STRs much longer than 100 bp, the typical length of short reads.We propose ab initio procedures for sensing and locating long STRs promptly by using the frequency distribution of all STRs and paired-end read information. We validated the reproducibility of this method using biological replicates and used it to locate an STR associated with a brain disease (SCA31). Subsequently, we sequenced this STR site in 11 SCA31 samples using SMRT(TM) sequencing (Pacific Biosciences), determined 2.3-3.1 kb sequences at nucleotide resolution and revealed that (TGGAA)- and (TAAAATAGAA)-repeat expansions determined the instability of the repeat expansions associated with SCA31. Our method could also identify common STRs, (AAAG)- and (AAAAG)-repeat expansions, which are remarkably expanded at four positions in an SCA31 sample. This is the first proposed method for rapidly finding disease-associated long STRs in personal genomes using hybrid sequencing of short and long reads.Our TRhist software is available at data are available at Bioinformatics online.

July 19, 2019  |  

Resolving the complexity of the human genome using single-molecule sequencing.

The human genome is arguably the most complete mammalian reference assembly, yet more than 160 euchromatic gaps remain and aspects of its structural variation remain poorly understood ten years after its completion. To identify missing sequence and genetic variation, here we sequence and analyse a haploid human genome (CHM1) using single-molecule, real-time DNA sequencing. We close or extend 55% of the remaining interstitial gaps in the human GRCh37 reference genome–78% of which carried long runs of degenerate short tandem repeats, often several kilobases in length, embedded within (G+C)-rich genomic regions. We resolve the complete sequence of 26,079 euchromatic structural variants at the base-pair level, including inversions, complex insertions and long tracts of tandem repeats. Most have not been previously reported, with the greatest increases in sensitivity occurring for events less than 5 kilobases in size. Compared to the human reference, we find a significant insertional bias (3:1) in regions corresponding to complex insertions and long short tandem repeats. Our results suggest a greater complexity of the human genome in the form of variation of longer and more complex repetitive DNA that can now be largely resolved with the application of this longer-read sequencing technology.

July 19, 2019  |  

Resolving complex tandem repeats with long reads.

Resolving tandemly repeated genomic sequences is a necessary step in improving our understanding of the human genome. Short tandem repeats (TRs), or microsatellites, are often used as molecular markers in genetics, and clinically, variation in microsatellites can lead to genetic disorders like Huntington’s diseases. Accurately resolving repeats, and in particular TRs, remains a challenging task in genome alignment, assembly and variation calling. Though tools have been developed for detecting microsatellites in short-read sequencing data, these are limited in the size and types of events they can resolve. Single-molecule sequencing technologies may potentially resolve a broader spectrum of TRs given their increased length, but require new approaches given their significantly higher raw error profiles. However, due to inherent error profiles of the single-molecule technologies, these reads presents a unique challenge in terms of accurately identifying and estimating the TRs.Here we present PacmonSTR, a reference-based probabilistic approach, to identify the TR region and estimate the number of these TR elements in long DNA reads. We present a multistep approach that requires as input, a reference region and the reference TR element. Initially, the TR region is identified from the long DNA reads via a 3-stage modified Smith-Waterman approach and then, expected number of TR elements is calculated using a pair-Hidden Markov Models-based method. Finally, TR-based genotype selection (or clustering: homozygous/heterozygous) is performed with Gaussian mixture models, using the Akaike information criteria, and coverage expectations. © The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please email:

July 19, 2019  |  

An incomplete understanding of human genetic variation.

Deciphering the genetic basis of human disease requires a comprehensive knowledge of genetic variants irrespective of their class or frequency. Although an impressive number of human genetic variants have been catalogued, a large fraction of the genetic difference that distinguishes two human genomes is still not understood at the base-pair level. This is because the emphasis has been on single-nucleotide variation as opposed to less tractable and more complex genetic variants, including indels and structural variants. The latter, we propose, will have a large impact on human phenotypes but require a more systematic assessment of genomes at deeper coverage and alternate sequencing and mapping technologies. Copyright © 2016 by the Genetics Society of America.

July 19, 2019  |  

Short tandem repeats, segmental duplications, gene deletion, and genomic instability in a rapidly diversified immune gene family.

Genomic regions with repetitive sequences are considered unstable and prone to swift DNA diversification processes. A highly diverse immune gene family of the sea urchin (Strongylocentrotus purpuratus), called Sp185/333, is composed of clustered genes with similar sequence as well as several types of repeats ranging in size from short tandem repeats (STRs) to large segmental duplications. This repetitive structure may have been the basis for the incorrect assembly of this gene family in the sea urchin genome sequence. Consequently, we have resolved the structure of the family and profiled the members by sequencing selected BAC clones using Illumina and PacBio approaches.BAC insert assemblies identified 15 predicted genes that are organized into three clusters. Two of the gene clusters have almost identical flanking regions, suggesting that they may be non-matching allelic clusters residing at the same genomic locus. GA STRs surround all genes and appear in large stretches at locations of putatively deleted genes. GAT STRs are positioned at the edges of segmental duplications that include a subset of the genes. The unique locations of the STRs suggest their involvement in gene deletions and segmental duplications. Genomic profiling of the Sp185/333 gene diversity in 10 sea urchins shows that no gene repertoires are shared among individuals indicating a very high gene diversification rate for this family.The repetitive genomic structure of the Sp185/333 family that includes STRs in strategic locations may serve as platform for a controlled mechanism which regulates the processes of gene recombination, gene conversion, duplication and deletion. The outcome is genomic instability and allelic mismatches, which may further drive the swift diversification of the Sp185/333 gene family that may improve the immune fitness of the species.

July 7, 2019  |  

A genomic view of short tandem repeats.

Short tandem repeats (STRs) are some of the fastest mutating loci in the genome. Tools for accurately profiling STRs from high-throughput sequencing data have enabled genome-wide interrogation of more than a million STRs across hundreds of individuals. These catalogs have revealed that STRs are highly multiallelic and may contribute more de novo mutations than any other variant class. Recent studies have leveraged these catalogs to show that STRs play a widespread role in regulating gene expression and other molecular phenotypes. These analyses suggest that STRs are an underappreciated but rich reservoir of variation that likely make significant contributions to Mendelian diseases, complex traits, and cancer. Copyright © 2017 Elsevier Ltd. All rights reserved.

July 7, 2019  |  

Short tandem repeat number estimation from paired-end reads for multiple individuals by considering coalescent tree.

Two types of approaches are mainly considered for the repeat number estimation in short tandem repeat (STR) regions from high-throughput sequencing data: approaches directly counting repeat patterns included in sequence reads spanning the region and approaches based on detecting the difference between the insert size inferred from aligned paired-end reads and the actual insert size. Although the accuracy of repeat numbers estimated with the former approaches is high, the size of target STR regions is limited to the length of sequence reads. On the other hand, the latter approaches can handle STR regions longer than the length of sequence reads. However, repeat numbers estimated with the latter approaches is less accurate than those with the former approaches.We proposed a new statistical model named coalescentSTR that estimates repeat numbers from paired-end read distances for multiple individuals simultaneously by connecting the read generative model for each individual with their genealogy. In the model, the genealogy is represented by handling coalescent trees as hidden variables, and the summation of the hidden variables is taken on coalescent trees sampled based on phased genotypes located around a target STR region with Markov chain Monte Carlo. In the sampled coalescent trees, repeat number information from insert size data is propagated, and more accurate estimation of repeat numbers is expected for STR regions longer than the length of sequence reads. For finding the repeat numbers maximizing the likelihood of the model on the estimation of repeat numbers, we proposed a state-of-the-art belief propagation algorithm on sampled coalescent trees.We verified the effectiveness of the proposed approach from the comparison with existing methods by using simulation datasets and real whole genome and whole exome data for HapMap individuals analyzed in the 1000 Genomes Project.

July 7, 2019  |  

STR-realigner: a realignment method for short tandem repeat regions.

In the estimation of repeat numbers in a short tandem repeat (STR) region from high-throughput sequencing data, two types of strategies are mainly taken: a strategy based on counting repeat patterns included in sequence reads spanning the region and a strategy based on estimating the difference between the actual insert size and the insert size inferred from paired-end reads. The quality of sequence alignment is crucial, especially in the former approaches although usual alignment methods have difficulty in STR regions due to insertions and deletions caused by the variations of repeat numbers.We proposed a new dynamic programming based realignment method named STR-realigner that considers repeat patterns in STR regions as prior knowledge. By allowing the size change of repeat patterns with low penalty in STR regions, accurate realignment is expected. For the performance evaluation, publicly available STR variant calling tools were applied to three types of aligned reads: synthetically generated sequencing reads aligned with BWA-MEM, those realigned with STR-realigner, those realigned with ReviSTER, and those realigned with GATK IndelRealigner. From the comparison of root mean squared errors between estimated and true STR region size, the results for the dataset realigned with STR-realigner are better than those for other cases. For real data analysis, we used a real sequencing dataset from Illumina HiSeq 2000 for a parent-offspring trio. RepeatSeq and lobSTR were applied to the sequence reads for these individuals aligned with BWA-MEM, those realigned with STR-realigner, ReviSTER, and GATK IndelRealigner. STR-realigner shows the best performance in terms of consistency of the size of estimated STR regions in Mendelian inheritance. Root mean squared error values were also calculated from the comparison of these estimated results with STR region sizes obtained from high coverage PacBio sequencing data, and the results from the realigned sequencing data with STR-realigner showed the least (the best) root mean squared error value.The effectiveness of the proposed realignment method for STR regions was verified from the comparison with an existing method on both simulation datasets and real whole genome sequencing dataset.

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