Long-read sequencing technology indicates genome-wide effects of non-B DNA on polymerization speed and error rate

Guiblet, Wilfried; Cremona, Marzia A.; Čechová, Monika; Harris, Robert S.; Kejnovská, Iva; Kejnovský, Eduard; Eckert, Kristin A.; Chiaromonte, Francesca; Makova, Kateryna D.

doi:10.1101/gr.241257.118

Cited by 63 publications

(71 citation statements)

References 72 publications

(95 reference statements)

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“…While Illumina reads always represent short fragmented DNA, long DNA molecules used for PacBio and Nanopore sequencing could form secondary structures. We have recently shown that non-B DNA structures can affect PacBio sequencing depth and error rates (Guiblet et al 2018) . For Nanopore, fragments harboring these structures might not pass through the pores.…”

Section: Interspecific Differences and Lack Of Phylogenetic Signal Inmentioning

confidence: 99%

High satellite repeat turnover in great apes studied with short- and long-read technologies

Čechová

Harris

Tomaszkiewicz

et al. 2018

Preprint

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Running title: High turnover of satellite DNA in great apes Abstract Satellite repeats are a structural component of centromeres and telomeres, and in some instances their divergence is known to drive speciation. Due to their highly repetitive nature, satellite sequences have been understudied and underrepresented in genome assemblies. To investigate their turnover in great apes, we studied satellite repeats of unit sizes up to 50 bp in human, chimpanzee, bonobo, gorilla, and Sumatran and Bornean orangutans, using unassembled short and long sequencing reads. The density of satellite repeats, as identified from accurate short reads (Illumina), varied greatly among great ape genomes. These were dominated by a handful of abundant repeated motifs, frequently shared among species, which formed two groups: (1) the (AATGG) n repeat (critical for heat shock response) and its derivatives; and (2) subtelomeric 32-mers involved in telomeric metabolism. Using the densities of abundant repeats, individuals could be classified into species. However clustering did not reproduce the accepted species phylogeny, suggesting rapid repeat evolution. Several abundant repeats were enriched in males vs. females; using Y chromosome assemblies or FIuorescent In Situ Hybridization, we validated their location on the Y. Finally, applying a novel computational tool, we identified many satellite repeats completely embedded within long Oxford Nanopore and Pacific Biosciences reads. Such repeats were up to 59 kb in length and consisted of perfect repeats interspersed with other similar sequences. Our results based on sequencing reads generated with three different technologies provide the first detailed characterization of great ape satellite repeats, and open new avenues for exploring their functions.

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Section: Interspecific Differences and Lack Of Phylogenetic Signal Inmentioning

confidence: 99%

High satellite repeat turnover in great apes studied with short- and long-read technologies

Čechová

Harris

Tomaszkiewicz

et al. 2018

Preprint

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“…However, it remains unclear if this applies to sequence fragments with > 60% GC content; specifically whether read depth sequence errors are within acceptable limits using PacBio technology. Furthermore, at the same time as this manuscript was reviewed another study was published [33] which evaluated the efficiency of PacBio reads for sequencing non-B DNA regions in human and mouse genomes. This study showed that non-B DNA regions modified the polymerization speed and the error rate, i.e.…”

Section: Resultsmentioning

confidence: 99%

“…regions able to determine non-B structures plus their single-stranded DNA environment due to transitions between B and non-B DNA) represent about 13% of the studied genomes [34, 35]. Among the seven kinds of non-B DNA determinants that were investigated, they showed [33] that mammalian regions containing stretches of direct repeats and were composed mostly of G-quadruplexes (G4; i.e. regions containing the motif G 3 + N1-12 G 3 + N 1-12 G 3 + N 1-12 G 3 where N is any base including G) had pronounced effects on read depth and the error rate.…”

Section: Resultsmentioning

confidence: 99%

“…With respect to sequence error rates, we observed that over the entire length of the CPT1C and RNL3 genes, it was close to that previously described when benchmarking this technology (error rate ranging from 11 to 15%, depending on the sequence; for review see [22]). This was in striking contrast to the rDNA and SV2A genes for which error rates were between 15 to 25% (because the sequences were GC rich, sequences with error rates above 25% could not be properly aligned [33]). Furthermore, we observed that only certain regions in long reads had an error rate below 25%.…”

Section: Resultsmentioning

confidence: 99%

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Sequence properties of certain GC rich avian genes, their origins and absence from genome assemblies: case studies

et al. 2019

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Background More and more eukaryotic genomes are sequenced and assembled, most of them presented as a complete model in which missing chromosomal regions are filled by Ns and where a few chromosomes may be lacking. Avian genomes often contain sequences with high GC content, which has been hypothesized to be at the origin of many missing sequences in these genomes. We investigated features of these missing sequences to discover why some may not have been integrated into genomic libraries and/or sequenced. Results The sequences of five red jungle fowl cDNA models with high GC content were used as queries to search publicly available datasets of Illumina and Pacbio sequencing reads. These were used to reconstruct the leptin, TNFα, MRPL52, PCP2 and PET100 genes, all of which are absent from the red jungle fowl genome model. These gene sequences displayed elevated GC contents, had intron sizes that were sometimes larger than non-avian orthologues, and had non-coding regions that contained numerous tandem and inverted repeat sequences with motifs able to assemble into stable G-quadruplexes and intrastrand dyadic structures. Our results suggest that Illumina technology was unable to sequence the non-coding regions of these genes. On the other hand, PacBio technology was able to sequence these regions, but with dramatically lower efficiency than would typically be expected. Conclusions High GC content was not the principal reason why numerous GC-rich regions of avian genomes are missing from genome assembly models. Instead, it is the presence of tandem repeats containing motifs capable of assembling into very stable secondary structures that is likely responsible.

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“…Several important discoveries have already been made from this kinetic information, such as the widespread presence of 6mA modifications in the human genome [6], a modification that was previously thought to only be present in bacterial genomes. In addition to base modifications, SMRT sequencing data also enables us to study other events, such as DNA conformations [7]. Another aspect of SMRT sequencing is that it can be used to study RNA, and it is currently the only technology that can generate high-quality continuous sequences for full-length transcripts up to 10 kb or more.…”

mentioning

confidence: 99%

The Versatility of SMRT Sequencing

Hestand

Ameur

2019

Genes

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Long-read sequencing technology indicates genome-wide effects of non-B DNA on polymerization speed and error rate

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Cited by 63 publications

References 72 publications

High satellite repeat turnover in great apes studied with short- and long-read technologies

High satellite repeat turnover in great apes studied with short- and long-read technologies

Sequence properties of certain GC rich avian genes, their origins and absence from genome assemblies: case studies

The Versatility of SMRT Sequencing

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