Insertion Bias Research Articles

The impact of insertion bias into piRNA clusters on the invasion of transposable elements.

This study challenges the pre-existing understanding of the TE dynamics by investigating the potential adaptive role of insertion bias into piRNA clusters. Using extensive forward simulations, we demonstrate that while insertion bias significantly alters TE invasion dynamics, it is generally not beneficial for the TE's spread in populations. Our results also show that transposable elements (TEs) that avoid piRNA clusters tend to do better than those that are more likely to preferencially insert into these clusters. This work provides novel insights into the complex dynamics between TEs and host genomes, showing the limited scenarios where the insertion bias could be advantageous to TEs. These results open new area for research into TE invasion dynamics and the evolution of host-TE interactions, further contributing to our understanding of genome evolution and stability.

Heterologous survey of 130 DNA transposons in human cells highlights their functional divergence and expands the genome engineering toolbox

Experimental studies on DNA transposable elements (TEs) have been limited in scale, leading to a lack of understanding of the factors influencing transposition activity, evolutionary dynamics, and application potential as genome engineering tools. We predicted 130 active DNA TEs from 102 metazoan genomes and evaluated their activity in human cells. We identified 40 active (integration-competent) TEs, surpassing the cumulative number (20) of TEs found previously. With this unified comparative data, we found that the Tc1/mariner superfamily exhibits elevated activity, potentially explaining their pervasive horizontal transfers. Further functional characterization of TEs revealed additional divergence in features such as insertion bias. Remarkably, in CAR-T therapy for hematological and solid tumors, Mariner2_AG (MAG), the most active DNA TE identified, largely outperformed two widely used vectors, the lentiviral vector and the TE-based vector SB100X. Overall, this study highlights the varied transposition features and evolutionary dynamics of DNA TEs and increases the TE toolbox diversity.

Abstract 7435: PATTY: A bias estimation and correction model for bulk and single-cell CUT&Tag data

Abstract The accurate detection of transcription factor (TF) binding sites and histone modifications (HM) on the genome-wide scale is essential for studying functional epigenetics and gene regulation. Cleavage Under Targets & Tagmentation (CUT&Tag) is a low-cost and easy-to-implement epigenomic profiling method that can be performed on a low number of cells and on the single-cell level. CUT&Tag experiments use the hyperactive transposase Tn5 for tagmentation. We find that Tn5 is subject to intrinsic sequence insertion bias (intrinsic bias). Additionally, the preference of Tn5 insertion toward accessible chromatin also affects the distribution of CUT&Tag reads (open chromatin bias). Both types of biases can significantly confound bulk and single-cell CUT&Tag data analysis, which requires careful assessment and new analytical methods. To address this challenge, we present PATTY (Propensity Analyzer for Tn5 Transposase Yielded bias), a computational method for systematic characterization and correction of biases in CUT&Tag data. We demonstrate that histone modification signals (H3K27me3 and H3K27ac) detected from CUT&Tag data after bias correction using PATTY are better associated with orthogonal biological features such as gene expression. PATTY-corrected single-cell CUT&Tag signals for histone modification can better cluster cells into their true cell types. This new computational method can improve both bulk and single-cell CUT&Tag data analysis. Citation Format: Sheng'en Shawn Hu, Qingying Chen, Megan Grieco, Chongzhi Zang. PATTY: A bias estimation and correction model for bulk and single-cell CUT&Tag data [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7435.

Interchromosomal Colocalization with Parental Genes Is Linked to the Function and Evolution of Mammalian Retrocopies.

Retrocopies are gene duplicates arising from reverse transcription of mature mRNA transcripts and their insertion back into the genome. While long being regarded as processed pseudogenes, more and more functional retrocopies have been discovered. How the stripped-down retrocopies recover expression capability and become functional paralogs continually intrigues evolutionary biologists. Here, we investigated the function and evolution of retrocopies in the context of 3D genome organization. By mapping retrocopy-parent pairs onto sequencing-based and imaging-based chromatin contact maps in human and mouse cell lines and onto Hi-C interaction maps in 5 other mammals, we found that retrocopies and their parental genes show a higher-than-expected interchromosomal colocalization frequency. The spatial interactions between retrocopies and parental genes occur frequently at loci in active subcompartments and near nuclear speckles. Accordingly, colocalized retrocopies are more actively transcribed and translated and are more evolutionarily conserved than noncolocalized ones. The active transcription of colocalized retrocopies may result from their permissive epigenetic environment and shared regulatory elements with parental genes. Population genetic analysis of retroposed gene copy number variants in human populations revealed that retrocopy insertions are not entirely random in regard to interchromosomal interactions and that colocalized retroposed gene copy number variants are more likely to reach high frequencies, suggesting that both insertion bias and natural selection contribute to the colocalization of retrocopy-parent pairs. Further dissection implies that reduced selection efficacy, rather than positive selection, contributes to the elevated allele frequency of colocalized retroposed gene copy number variants. Overall, our results hint a role of interchromosomal colocalization in the "resurrection" of initially neutral retrocopies.

The genomic distribution of transposable elements is driven by spatially variable purifying selection.

It is widely accepted that the genomic distribution of transposable elements (TEs) mainly reflects the outcome of purifying selection and insertion bias (1). Nevertheless, the relative importance of these two evolutionary forces could not be tested thoroughly. Here, we introduce an experimental system, which allows separating purifying selection from TE insertion bias. We used experimental evolution to study the TE insertion patterns in Drosophila simulans founder populations harboring 1040 insertions of an active P-element. After 10 generations at a large population size, we detected strong selection against P-element insertions. The exception were P-element insertions in genomic regions for which a strong insertion bias has been proposed (2-4). Because recurrent P-element insertions cannot explain this pattern, we conclude that purifying selection, with variable strength along the chromosomes, is the major determinant of the genomic distribution of P-elements. Genomic regions with relaxed purifying selection against P-element insertions exhibit normal levels of purifying selection against base substitutions. This suggests that different types of purifying selection operate on base substitutions and P-element insertions. Our results highlight the power of experimental evolution to understand basic evolutionary processes, which are difficult to infer from patterns of natural variation alone.

Genetic basis of I-complex plasmid stability and conjugation.

Plasmids are major drivers of increasing antibiotic resistance, necessitating an urgent need to understand their biology. Here we describe a detailed dissection of the molecular components controlling the genetics of I-complex plasmids, a group of antibiotic resistance plasmids found frequently in pathogenic Escherichia coli and other Enterobacteriaceae that cause significant human disease. We show these plasmids cluster into four distinct subgroups, with the prototype IncI1 plasmid R64 subgroup displaying low nucleotide sequence conservation to other I-complex plasmids. Using pMS7163B, an I-complex plasmid distantly related to R64, we performed a high-resolution transposon-based genetic screen and defined genes involved in replication, stability, and conjugative transfer. We identified the replicon and a partitioning system as essential for replication/stability. Genes required for conjugation included the type IV secretion system, relaxosome, and several uncharacterised genes located in the pMS7163B leading transfer region that exhibited an upstream strand-specific transposon insertion bias. The overexpression of these genes severely impacted host cell growth or reduced fitness during mixed competitive growth, demonstrating that their expression must be controlled to avoid deleterious impacts. These genes were present in >80% of all I-complex plasmids and broadly conserved across multiple plasmid incompatibility groups, implicating an important role in plasmid dissemination.

The evolutionary dynamics that retain long neutral genomic sequences in face of indel deletion bias: a model and its application to human introns.

Insertions and deletions (indels) of short DNA segments are common evolutionary events. Numerous studies showed that deletions occur more often than insertions in both prokaryotes and eukaryotes. It raises the question why neutral sequences are not eradicated from the genome. We suggest that this is due to a phenomenon we term border-induced selection. Accordingly, a neutral sequence is bordered between conserved regions. Deletions occurring near the borders occasionally protrude to the conserved region and are thereby subject to strong purifying selection. Thus, for short neutral sequences, an insertion bias is expected. Here, we develop a set of increasingly complex models of indel dynamics that incorporate border-induced selection. Furthermore, we show that short conserved sequences within the neutrally evolving sequence help explain: (i) the presence of very long sequences; (ii) the high variance of sequence lengths; and (iii) the possible emergence of multimodality in sequence length distributions. Finally, we fitted our models to the human intron length distribution, as introns are thought to be mostly neutral and bordered by conserved exons. We show that when accounting for the occurrence of short conserved sequences within introns, we reproduce the main features, including the presence of long introns and the multimodality of intron distribution.

An Ancient Clade of Penelope-Like Retroelements with Permuted Domains Is Present in the Green Lineage and Protists, and Dominates Many Invertebrate Genomes.

Penelope-like elements (PLEs) are an enigmatic clade of retrotransposons whose reverse transcriptases (RTs) share a most recent common ancestor with telomerase RTs. The single ORF of canonical endonuclease (EN)+ PLEs encodes RT and a C-terminal GIY–YIG EN that enables intrachromosomal integration, whereas EN− PLEs lack EN and are generally restricted to chromosome termini. EN+ PLEs have only been found in animals, except for one case of horizontal transfer to conifers, whereas EN− PLEs occur in several kingdoms. Here, we report a new, deep-branching PLE clade with a permuted domain order, whereby an N-terminal GIY–YIG EN is linked to a C-terminal RT by a short domain with a characteristic CxC motif. These N-terminal EN+ PLEs share a structural organization, including pseudo-LTRs and complex tandem/inverted insertions, with canonical EN+ PLEs from Penelope/Poseidon, Neptune, and Nematis clades, and show insertion bias for microsatellites, but lack canonical hammerhead ribozyme motifs. However, their phylogenetic distribution is much broader. The Naiads, found in numerous invertebrate phyla, can reach tens of thousands of copies per genome. In spiders and clams, Naiads independently evolved to encode selenoproteins containing multiple selenocysteines. Chlamys, which lack the CCHH motif universal to PLE ENs, occur in green algae, spike mosses (targeting ribosomal DNA), and slime molds. Unlike canonical PLEs, RTs of N-terminal EN+ PLEs contain the insertion-in-fingers domain (IFD), strengthening the link between PLEs and telomerases. Additionally, we describe Hydra, a novel metazoan C-terminal EN+ clade. Overall, we conclude that PLE diversity, taxonomic distribution, and abundance are comparable with non-LTR and LTR-retrotransposons.

Calculation Methods of Solution Chemical Potential and Application in Emulsion Microencapsulation.

Several new biased sampling methods were summarized for solution chemical potential calculation methods in the field of emulsion microencapsulation. The principles, features, and calculation efficiencies of various biased Widom insertion sampling methods were introduced, including volume detection bias, simulation ensemble bias, and particle insertion bias. The proper matches between various types of solution in emulsion and biased Widom methods were suggested, following detailed analyses on the biased insertion techniques. The volume detection bias methods effectively improved the accuracy of the data and the calculation efficiency by inserting detection particles and were suggested to be used for the calculation of solvent chemical potential for the homogeneous aqueous phase of the emulsion. The chemical potential of water, argon, and fluorobenzene (a typical solvent of the oil phase in double emulsion) was calculated by a new, optimized volume detection bias proposed by this work. The recently developed Well-Tempered(WT)-Metadynamics method skillfully constructed low-density regions for particle insertion and dynamically adjusted the system configuration according to the potential energy around the detection point, and hence, could be used for the oil-polymer mixtures of microencapsulation emulsion. For the macromolecule solutes in the oil or aqueous phase of the emulsion, the particle insertion bias could be applied to greatly increase the success rate of Widom insertions. Readers were expected to choose appropriate biased Widom methods to carry out their calculations on chemical potential, fugacity, and solubility of solutions based on the system molecular properties, inspired by this paper.

Fundamental asymmetry of insertions and deletions in genomes size evolution

The origin of large genomes that underlies the long standing “C-value enigma” is only partially explained by selfish DNA. We investigated insertions and deletions (indels) of nucleotides and discussed their relevance in size evolution of random biological sequences (RBS) and genomes. By developing a probabilistic model of RBS based on size evolution of expandable sites in a thought perfect genome, it was found that insertion bias engenders exponential increase of average RBS sizes. When combined with existing large segments of genome that are not subject to selection pressure (e.g. selfish DNA), such insertion bias results in explosive expansion of genomes, and therefore helps explain the “C value enigma” besides selfish DNA. Such increase of RBS size is caused by the fundamental asymmetry of indels, with insertions result in more available sites and deletions result in less deletable nucleotides. In qualitative agreement with the size distribution of known genomes, tails of RBS size distributions exhibit exponential decay with probabilities of larger RBS segments being smaller. Unsurprisingly, a slight deletion bias (higher deletions probabilities) results in a slow decrease of average RBS size and may lead to their eventual vanishing. Contrary to intuition, strictly balanced insertion and deletion results in linearly increasing instead of completely fixed RBS size. Nonetheless, such slow linear increase of average RBS sizes with time are small in magnitude and are consequently not influential on genome size evolution, and certainly not a major contributor for the “C-value enigma”. Our model suggested that insertion bias of nucleotides may provide complementary explanation for large genomes besides selfish DNA. The fundamental indel asymmetry is applicable for all forms of genomic insertions and deletions. Long-lasting exponential increase of genome size present energy and material requirement that is impossible to sustain. We therefore concluded that if there were explosively accelerating expansion caused by significant effective insertion bias for any survival species, it must have occurred sporadically. Our model also provided an explanation for the observed proportional evolution of genome size.

The impact of poly-A microsatellite heterologies in meiotic recombination.

Meiotic recombination has strong, but poorly understood effects on short tandem repeat (STR) instability. Here, we screened thousands of single recombinant products with sperm typing to characterize the role of polymorphic poly-A repeats at a human recombination hotspot in terms of hotspot activity and STR evolution. We show that the length asymmetry between heterozygous poly-A's strongly influences the recombination outcome: a heterology of 10 A's (9A/19A) reduces the number of crossovers and elevates the frequency of non-crossovers, complex recombination products, and long conversion tracts. Moreover, the length of the heterology also influences the STR transmission during meiotic repair with a strong and significant insertion bias for the short heterology (6A/7A) and a deletion bias for the long heterology (9A/19A). In spite of this opposing insertion-/deletion-biased gene conversion, we find that poly-A's are enriched at human recombination hotspots that could have important consequences in hotspot activation.

Foamy Virus Vector Carries a Strong Insulator in Its Long Terminal Repeat Which Reduces Its Genotoxic Potential

Strong viral enhancers in gammaretrovirus vectors have caused cellular proto-oncogene activation and leukemia, necessitating the use of cellular promoters in "enhancerless" self-inactivating integrating vectors. However, cellular promoters result in relatively low transgene expression, often leading to inadequate disease phenotype correction. Vectors derived from foamy virus, a nonpathogenic retrovirus, show higher preference for nongenic integrations than gammaretroviruses/lentiviruses and preferential integration near transcriptional start sites, like gammaretroviruses. We found that strong viral enhancers/promoters placed in foamy viral vectors caused extremely low immortalization of primary mouse hematopoietic stem/progenitor cells compared to analogous gammaretrovirus/lentivirus vectors carrying the same enhancers/promoters, an effect not explained solely by foamy virus' modest insertional site preference for nongenic regions compared to gammaretrovirus/lentivirus vectors. Using CRISPR/Cas9-mediated targeted insertion of analogous proviral sequences into the LMO2 gene and then measuring LMO2 expression, we demonstrate a sequence-specific effect of foamy virus, independent of insertional bias, contributing to reduced genotoxicity. We show that this effect is mediated by a 36-bp insulator located in the foamy virus long terminal repeat (LTR) that has high-affinity binding to the CCCTC-binding factor. Using our LMO2 activation assay, LMO2 expression was significantly increased when this insulator was removed from foamy virus and significantly reduced when the insulator was inserted into the lentiviral LTR. Our results elucidate a mechanism underlying the low genotoxicity of foamy virus, identify a novel insulator, and support the use of foamy virus as a vector for gene therapy, especially when strong enhancers/promoters are required.IMPORTANCE Understanding the genotoxic potential of viral vectors is important in designing safe and efficacious vectors for gene therapy. Self-inactivating vectors devoid of viral long-terminal-repeat enhancers have proven safe; however, transgene expression from cellular promoters is often insufficient for full phenotypic correction. Foamy virus is an attractive vector for gene therapy. We found foamy virus vectors to be remarkably less genotoxic, well below what was expected from their integration site preferences. We demonstrate that the foamy virus long terminal repeats contain an insulator element that binds CCCTC-binding factor and reduces its insertional genotoxicity. Our study elucidates a mechanism behind the low genotoxic potential of foamy virus, identifies a unique insulator, and supports the use of foamy virus as a vector for gene therapy.

Creation of a Dense Transposon Insertion Library Using Bacterial Conjugation in Enterobacterial Strains Such As<em> Escherichia Coli</em> or <em>Shigella flexneri</em>

Transposon mutagenesis is a method that allows gene disruption via the random genomic insertion of a piece of DNA called a transposon. The protocol below outlines a method for high efficiency transfer between bacterial strains of a plasmid harboring a transposon containing a kanamycin resistance marker. The plasmid-borne transposase is encoded by a variant tnp gene that inserts the transposon into the genome of the recipient strain with very low insertional bias. This method thus allows the creation of large mutant libraries in which transposons have been inserted into unique genomic positions in a recipient strain of either Escherichia coli or Shigella flexneri bacteria. By using bacterial conjugation, as opposed to other methods such as electroporation or chemical transformation, large libraries with hundreds of thousands of unique clones can be created. This yields high-density insertion libraries, with insertions occurring as frequently as every 4-6 base pairs in non-essential genes. This method is superior to other methods as it allows for an inexpensive, easy to use, and high efficiency method for the creation of a dense transposon insertion library. The transposon library can be used in downstream applications such as transposon sequencing (Tn-Seq), to infer genetic interaction networks, or more simply, in mutational (forward genetic) screens.

Creation of a Dense Transposon Insertion Library Using Bacterial Conjugation in Enterobacterial Strains Such As Escherichia Coli or Shigella flexneri.

Transposon mutagenesis is a method that allows gene disruption via the random genomic insertion of a piece of DNA called a transposon. The protocol below outlines a method for high efficiency transfer between bacterial strains of a plasmid harboring a transposon containing a kanamycin resistance marker. The plasmid-borne transposase is encoded by a variant tnp gene that inserts the transposon into the genome of the recipient strain with very low insertional bias. This method thus allows the creation of large mutant libraries in which transposons have been inserted into unique genomic positions in a recipient strain of either Escherichia coli or Shigella flexneri bacteria. By using bacterial conjugation, as opposed to other methods such as electroporation or chemical transformation, large libraries with hundreds of thousands of unique clones can be created. This yields high-density insertion libraries, with insertions occurring as frequently as every 4-6 base pairs in non-essential genes. This method is superior to other methods as it allows for an inexpensive, easy to use, and high efficiency method for the creation of a dense transposon insertion library. The transposon library can be used in downstream applications such as transposon sequencing (Tn-Seq), to infer genetic interaction networks, or more simply, in mutational (forward genetic) screens.

GINOM: A statistical framework for assessing interval overlap of multiple genomic features.

A common problem in genomics is to test for associations between two or more genomic features, typically represented as intervals interspersed across the genome. Existing methodologies can test for significant pairwise associations between two genomic intervals; however, they cannot test for associations involving multiple sets of intervals. This limits our ability to uncover more complex, yet biologically important associations between multiple sets of genomic features. We introduce GINOM (Genomic INterval Overlap Model), a new method that enables testing of significant associations between multiple genomic features. We demonstrate GINOM’s ability to identify higher-order associations with both simulated and real data. In particular, we used GINOM to explore L1 retrotransposable element insertion bias in lung cancer and found a significant pairwise association between L1 insertions and heterochromatic marks. Unlike other methods, GINOM also detected an association between L1 insertions and gene bodies marked by a facultative heterochromatic mark, which could explain the observed bias for L1 insertions towards cancer-associated genes.

Comprehensive Essentiality Analysis of the Mycobacterium tuberculosis Genome via Saturating Transposon Mutagenesis.

ABSTRACT For decades, identifying the regions of a bacterial chromosome that are necessary for viability has relied on mapping integration sites in libraries of random transposon mutants to find loci that are unable to sustain insertion. To date, these studies have analyzed subsaturated libraries, necessitating the application of statistical methods to estimate the likelihood that a gap in transposon coverage is the result of biological selection and not the stochasticity of insertion. As a result, the essentiality of many genomic features, particularly small ones, could not be reliably assessed. We sought to overcome this limitation by creating a completely saturated transposon library in Mycobacterium tuberculosis. In assessing the composition of this highly saturated library by deep sequencing, we discovered that a previously unknown sequence bias of the Himar1 element rendered approximately 9% of potential TA dinucleotide insertion sites less permissible for insertion. We used a hidden Markov model of essentiality that accounted for this unanticipated bias, allowing us to confidently evaluate the essentiality of features that contained as few as 2 TA sites, including open reading frames (ORF), experimentally identified noncoding RNAs, methylation sites, and promoters. In addition, several essential regions that did not correspond to known features were identified, suggesting uncharacterized functions that are necessary for growth. This work provides an authoritative catalog of essential regions of the M. tuberculosis genome and a statistical framework for applying saturating mutagenesis to other bacteria.

Improved genome sequencing using an engineered transposase

BackgroundNext-generation sequencing (NGS) has transformed genomic research by reducing turnaround time and cost. However, no major breakthrough has been made in the upstream library preparation methods until the transposase-based Nextera method was invented. Nextera combines DNA fragmentation and barcoding in a single tube reaction and therefore enables a very fast workflow to sequencing-ready DNA libraries within a couple of hours. When compared to the traditional ligation-based methods, transposed-based Nextera has a slight insertion bias.ResultsHere we present the discovery of a mutant transposase (Tn5-059) with a lowered GC insertion bias through protein engineering. We demonstrate Tn5-059 reduces AT dropout and increases uniformity of genome coverage in both bacterial genomes and human genome. We also observe higher library diversity generated by Tn5-059 when compared to Nextera v2 for human exomes, which leads to less sequencing and lower cost per genome. In addition, when used for human exomes, Tn5-059 delivers consistent library insert size over a range of input DNA, allowing up to a tenfold variance from the 50 ng input recommendation.ConclusionsEnhanced DNA input tolerance of Tn5-059 can translate to flexibility and robustness of workflow. DNA input tolerance together with superior uniformity of coverage and lower AT dropouts extend the applications of transposase based library preps. We discuss possible mechanisms of improvements in Tn5-059, and potential advantages of using the new mutant in varieties of applications including microbiome sequencing and chromatin profiling.

Genome-Wide Estimates of Mutation Rates and Spectrum in Schizosaccharomyces pombe Indicate CpG Sites are Highly Mutagenic Despite the Absence of DNA Methylation.

We accumulated mutations for 1952 generations in 79 initially identical, haploid lines of the fission yeast Schizosaccharomyces pombe, and then performed whole-genome sequencing to determine the mutation rates and spectrum. We captured 696 spontaneous mutations across the 79 mutation accumulation (MA) lines. We compared the mutation spectrum and rate to a recently published equivalent experiment on the same species, and to another model ascomycetous yeast, the budding yeast Saccharomyces cerevisiae. While the two species are approximately 600 million years diverged from each other, they share similar life histories, genome size and genomic G/C content. We found that Sc. pombe and S. cerevisiae have similar mutation rates, but Sc. pombe exhibits a stronger insertion bias. Intriguingly, we observed an increased mutation rate at cytosine nucleotides, specifically CpG nucleotides, which is also seen in S. cerevisiae. However, the absence of methylation in Sc. pombe and the pattern of mutation at these sites, primarily C → A as opposed to C → T, strongly suggest that the increased mutation rate is not caused by deamination of methylated cytosines. This result implies that the high mutability of CpG dinucleotides in other species may be caused in part by a methylation-independent mechanism. Many of our findings mirror those seen in the recent study, despite the use of different passaging conditions, indicating that MA is a reliable method for estimating mutation rates and spectra.

Impact of three Illumina library construction methods on GC bias and HLA genotype calling

Next-generation sequencing (NGS) is increasingly recognized for its ability to overcome allele ambiguity and deliver high-resolution typing in the HLA system. Using this technology, non-uniform read distribution can impede the reliability of variant detection, which renders high-confidence genotype calling particularly difficult to achieve in the polymorphic HLA complex. Recently, library construction has been implicated as the dominant factor in instigating coverage bias. To study the impact of this phenomenon on HLA genotyping, we performed long-range PCR on 12 samples to amplify HLA-A, -B, -C, -DRB1, and -DQB1, and compared the relative contribution of three Illumina library construction methods (TruSeq Nano, Nextera, Nextera XT) in generating downstream bias. Here, we show high GC% to be a good predictor of low sequencing depth. Compared to standard TruSeq Nano, GC bias was more prominent in transposase-based protocols, particularly Nextera XT, likely through a combination of transposase insertion bias being coupled with a high number of PCR enrichment cycles. Importantly, our findings demonstrate non-uniform read depth can have a direct and negative impact on the robustness of HLA genotyping, which has clinical implications for users when choosing a library construction strategy that aims to balance cost and throughput with data quality.

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.