Single-cell copy number variant detection reveals the dynamics and diversity of adaptation

Lauer, Stephanie; Avecilla, Grace; Spealman, Pieter; Sethia, Gunjan; Brandt, Nathan; Levy, Sasha F.; Gresham, David

doi:10.1101/381590

Cited by 10 publications

(15 citation statements)

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“…Inverted repeat sequences are CNVs that feature repeated sequences oriented in the opposite directions with a junction region separating them ( Figure 1b ). We recently identified numerous CNVs containing inverted repeat sequences ( Figure 1a ) in experimentally evolved strains of Saccharomyces cerevisiae using short-read Illumina sequencing [ 14 ] . These CNVs are likely formed by origin dependent inverted repeat amplification (ODIRA), a DNA replication-based mechanism of CNV formation [ 13 , 17 ].…”

Section: Resultsmentioning

confidence: 99%

“…All yeast strains were grown to greater than 1 x 10 7 cells/mL in 30mL minimal media [ 14 ] or YPD ( Supplemental Table 1 ). Genomic DNA (gDNA) from each strain was extracted using Qiagen 20/G Genomic tips from ~1.5x10 9 cells using the manufacturer's protocol.…”

Section: Library Preparationmentioning

confidence: 99%

“…This example shows a single read spanning a known CNV containing a triplication that comprises two inverted duplications ( A ). The hypothesized structure based on the alignment of Illumina sequencing reads to the reference genome [ 14 ] ( B ). The predicted inverted repeat junctions [ 14 ] are denoted with two vertical red lines.…”

Section: Data Availabilitymentioning

confidence: 99%

“…The hypothesized structure based on the alignment of Illumina sequencing reads to the reference genome [ 14 ] ( B ). The predicted inverted repeat junctions [ 14 ] are denoted with two vertical red lines. A rolling 1000 base window median phred score is calculated for the read ( C ).…”

Section: Data Availabilitymentioning

confidence: 99%

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Nanopore sequencing undergoes catastrophic sequence failure at inverted duplicated DNA sequences

Spealman

Burrell

Gresham

2019

Preprint

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1 Inverted duplicated sequences are a common feature of structural variants (SVs) and copy number variants (CNVs). Analysis of CNVs containing inverted duplicated sequences using nanopore sequencing identified recurrent aberrant behavior characterized by incorrect and low confidence base calls that result from a systematic elevation in the current recorded by the sequencing pore. The coincidence of inverted duplicated sequences with catastrophic sequence failure suggests that secondary DNA structures may impair transit through the nanopore.Advances in DNA sequencing have led to a greater understanding of the role structural variants play in disease [ 1 , 2 , 3 ] and evolution [ 4 ]. However, high throughput short read DNA sequencing faces intrinsic challenges in identifying complex structural variants. A variety of computational methods use sequence read depth, split, and discordant reads to identify structural variants. However, these methods are uninformative in low complexity and repetitive regions of the genome [ 5 , 2 , 6 ]. Long-read DNA sequencing can potentially overcome these technical limitations by generating reads of 10-100 kilobases (kb) length that enable unambiguous resolution of contiguous DNA sequences [ 7 , 8 , 9 ]. Nanopore sequencing, developed by Oxford-Nanopore Technologies (ONT), comprises a sequencing pore that translocates DNA or RNA across a membrane and sensors that measure changes in ionic current during the translocation.The electrical signals are subsequently converted into base-called sequences using a machine-learning base-caller [ 8 , 10 ]. The very long sequence reads that can be generated using this technology has proven invaluable for identifying many types of structural variants (SVs), such as translocations, tandem duplications, and simple inversions which are difficult to identify using short-read sequencing alone [ 11 , 12 ].Inverted duplications are a class of copy-number variants (CNVs) that feature two duplicated regions oriented in opposite directions with a junction between them. Inverted repeated DNA sequences occur frequently in many genomes. They have been identified as playing a role in adaptation and evolution [ 13 , 14 ] and have been associated with human pathologies and diseases [ 15 , 16 ].Here, we show that genomic regions containing large inverted duplications generate nanopore reads with pronounced base-calling errors and low sequence quality (i.e. phred scores). We propose that this high error rate is caused by biophysical interference induced upon the translocating DNA by the formation of large-scale secondary structures at inverted repeat sequences. As the specific secondary structures formed by these sequences are likely to depend on the length and nucleotide composition of the inverted repeat, it may be impossible to learn a generalizable signal for these sequences, presenting an intractable problem for nanopore sequencing.2

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Section: Resultsmentioning

confidence: 99%

Section: Library Preparationmentioning

confidence: 99%

Section: Data Availabilitymentioning

confidence: 99%

Section: Data Availabilitymentioning

confidence: 99%

See 2 more Smart Citations

Nanopore sequencing undergoes catastrophic sequence failure at inverted duplicated DNA sequences

Spealman

Burrell

Gresham

2019

Preprint

Self Cite

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“…For this reason, genetic variants must be present in a high frequency (i.e. ~50%) 59 in the population to be detected [23][24][25]. Sequencing at very high depth improves the detection 60 of low frequency CNVs, but the sensitivity is limited to large-scale CNVs present in > 5% cells 61 [26][27][28].…”

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confidence: 99%

Single cell sequencing of the small and AT-skewed genome of malaria parasites

Liu

Huckaby

Brown

et al. 2020

Preprint

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24Single cell genomics is a rapidly advancing field; however, most techniques are designed for 25 mammalian cells. Here, we present a single cell sequencing pipeline for the intracellular parasite, 26 Plasmodium falciparum, which harbors a relatively small genome with an extremely skewed 27 base content. Through optimization of a quasi-linear genome amplification method, we achieve 28 more even genome coverage and better targeting of the parasite genome over contaminants. 29 These improvements are particularly important for expanding the accessibility of single cell 30 approaches to new organisms and cell types and for improving the study of genetic mechanisms 31 of adaptation. 32 33 Keywords: whole-genome amplification, AT-skewed genome, malaria, single cell 34 sequencing, MALBAC 35 36 Background 37 Malaria is a life-threatening disease caused by protozoan Plasmodium parasites. P. falciparum 38 causes the greatest number of human malaria deaths [1]. The clinical symptoms of malaria occur 39 when parasites invade human erythrocytes and undergo rounds of asexual reproduction by 40 maturing from early forms into late stage parasites and bursting from erythrocytes to begin the 41 cycle again [2]. In this asexual cycle, parasites possess a single haploid genome during the early 42 stages; rapid genome replication in the later stages leads to an average of 16 genome copies [2].43 44Due to a lack of an effective vaccine, antimalarial drugs are required to treat malaria. However, 45 drug efficacy is threatened by the frequent emergence of resistant populations [3]. Copy number variations (CNVs), or the amplification and deletion of a genomic element, is one of the major 47 sources of genomic variation in P. falciparum that contribute to antimalarial resistance [4][5][6][7][8][9][10][11][12][13][14][15]. 48Similar to bacteria and viruses [16][17][18], a high rate of CNVs may initiate genomic changes that 49 contribute to the rapid adaptation of this organism [7,19]. Despite the importance of CNVs, their 50 dynamics in evolving populations are not well understood. 52The majority of CNVs in P. falciparum have been identified by analyzing bulk DNA in which 53 the CNVs are present in a substantial fraction of individual parasites in the population due to 54 positive selection [8,10,11,20,21]. However, many CNVs likely remain undetected because 55 they are presumably either deleterious or offer no advantages for parasite growth or transmission 56 [22] and are therefore present in low frequency [20,22]. Currently, most CNVs are identified 57 using read-depth analysis of short read sequencing data, which derives an average signal across 58 the population. For this reason, genetic variants must be present in a high frequency (i.e. ~50%) 59 in the population to be detected [23][24][25]. Sequencing at very high depth improves the detection 60 of low frequency CNVs, but the sensitivity is limited to large-scale CNVs present in > 5% cells 61 [26][27][28]. Analysis methods that rely on the detection of reads that span CNV junctions h...

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Temperature preference biases parental genome retention during hybrid evolution

Heil

Large

Patterson

et al. 2018

Preprint

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9Interspecific hybridization can introduce genetic variation that aids in adaptation to new or 10 changing environments. Here we investigate how the environment, and more specifically 11 temperature, interacts with hybrid genomes to alter parental genome representation over time. 12 We evolved Saccharomyces cerevisiae x Saccharomyces uvarum hybrids in nutrient-limited 13 continuous culture at 15°C for 200 generations. In comparison to previous evolution experiments 14 at 30°C, we identified a number of temperature specific responses, including the loss of the S. 15 cerevisiae allele in favor of the cryotolerant S. uvarum allele for several portions of the hybrid 16 genome. In particular, we discovered a genotype by environment interaction in the form of a 17 reciprocal loss of heterozygosity event on chromosome XIII. Which species haplotype is lost or 18 maintained is dependent on the parental species temperature preference and the temperature at 19 which the hybrid was evolved. We show that a large contribution to this directionality is due to 20 temperature sensitivity at a single locus, the high affinity phosphate transporter PHO84. This 21 work helps shape our understanding of what forces impact genome evolution after hybridization, 22 and how environmental conditions may favor or disfavor hybrids over time. 23 24 uvarum strains show evidence of introgression from several other yeast species, and S. cerevisiae 71x S. uvarum hybrids have been isolated from fermentation environments [55,59]. 72 We previously evolved S. cerevisiae x S. uvarum hybrids in the laboratory in several 73 nutrient-limited environments at the preferred growth temperature of S. cerevisiae [60]. We 74 frequently observed a phenomenon known as loss of heterozygosity (LOH) in these evolved 75 hybrids, in which an allele from one species is lost while the other species' allele is maintained. 76The outcome of such events is the homogenization of the hybrid genome at certain loci, and 77 represents a way in which a hybrid genome may become biased toward one parent's sub-78 genome. This type of mutation can occur due to gene conversion or break induced repair, and as 79 previously noted, has also been observed in organisms including S. pastorianus, pathogenic 80 hybrid yeast, and hybrid plants, but its role in adaptation has been unclear [47,61,62]. We used 81 genetic manipulation and competitive fitness assays to show that a particular set of LOH events 82 was the result of selection on the loss of the S. uvarum allele and amplification of the S. 83 cerevisiae allele at the high affinity phosphate transporter PHO84 in phosphate limited 84 conditions. By empirically demonstrating that LOH can be the product of selection, we 85 illuminated how an underappreciated mutation class can underlie adaptive hybrid phenotypes. 86This prior study illuminated how the environment (differences in nutrient availability) 87 can bias a hybrid genome towards one parent sub-genome. Due to many examples of genotype 88 by temperature interactio...

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Single-cell copy number variant detection reveals the dynamics and diversity of adaptation

Cited by 10 publications

References 97 publications

Nanopore sequencing undergoes catastrophic sequence failure at inverted duplicated DNA sequences

Nanopore sequencing undergoes catastrophic sequence failure at inverted duplicated DNA sequences

Single cell sequencing of the small and AT-skewed genome of malaria parasites

Temperature preference biases parental genome retention during hybrid evolution

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