BS-Seeker2: a versatile aligning pipeline for bisulfite sequencing data

Guo, Weilong; Fiziev, Petko; Wang, Yan; Cokus, Shawn J.; Sun, Xueqing; Zhang, Michael Q.; Chen, Pao‐Yang; Pellegrini, Matteo

doi:10.1186/1471-2164-14-774

Cited by 369 publications

(314 citation statements)

References 19 publications

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“…5A). This kind of distribution has been previously observed after bisulfite treatment of nonmethylated phage DNA (Guo et al 2013). Although phage reads with few cytosines were proposed to originate from random bisulfite conversion failure or sequencing errors, phage reads with high content of cytosines were thought to arise from secondary structure formation and overall failure of bisulfite conversion (Guo et al 2013).…”

Section: Arabidopsis Telomeres Are Unmethylatedsupporting

confidence: 57%

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Novel features of telomere biology revealed by the absence of telomeric DNA methylation

et al. 2016

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Cytosine methylation regulates the length and stability of telomeres, which can affect a wide variety of biological features, including cell differentiation, development, or illness. Although it is well established that subtelomeric regions are methylated, the presence of methylated cytosines at telomeres has remained controversial. Here, we have analyzed multiple bisulfite sequencing studies to address the methylation status of Arabidopsis thaliana telomeres. We found that the levels of estimated telomeric DNA methylation varied among studies. Interestingly, we estimated higher levels of telomeric DNA methylation in studies that produced C-rich telomeric strands with lower efficiency. However, these high methylation estimates arose due to experimental limitations of the bisulfite technique. We found a similar phenomenon for mitochondrial DNA: The levels of mitochondrial DNA methylation detected were higher in experiments with lower mitochondrial read production efficiencies. Based on experiments with high telomeric C-rich strand production efficiencies, we concluded that Arabidopsis telomeres are not methylated, which was confirmed by methylation-dependent restriction enzyme analyses. Thus, our studies indicate that telomeres are refractory to de novo DNA methylation by the RNA-directed DNA methylation machinery. This result, together with previously reported data, reveals that subtelomeric DNA methylation controls the homeostasis of telomere length.[Supplemental material is available for this article.]Telomeres guarantee the complete replication of chromosomal termini, prevent genome instability, and influence relevant systemic processes like aging, cancer, or illness (Blackburn 2010). The length of telomeres and the chromatin organization of telomeric regions influence telomere functions. Hence, the epigenetic marks that label telomeric regions, which include telomeres and subtelomeres, play important roles in telomere biology (Blasco 2007;Galati et al. 2013;Giraud-Panis et al. 2013).One of the major epigenetic signatures found in eukaryotes is cytosine methylation. This DNA modification regulates multiple processes in plants and animals, including the homeostasis of telomere length (Blasco 2007;Suzuki and Bird 2008;Ooi et al. 2009;Law and Jacobsen 2010;Castel and Martienssen 2013;Ogrocká et al. 2014;Vaquero-Sedas and Vega-Palas 2014). Mammalian DNA methylation is primarily found in the CG context (Ramsahoye et al. 2000;Lister et al. 2009). In contrast, plants have significant levels of DNA methylation in all sequence contexts (CG, CHG, and CHH, where H can be A, C, or T) (Law and Jacobsen 2010).Although subtelomeric DNA methylation has been reported in animals and plants, the presence of DNA methylation at telomeres remains an open question in both kingdoms (Blasco 2007;Vrbsky et al. 2010;Vaquero-Sedas et al. 2011;Ogrocká et al. 2014). The methylation status of mammalian telomeres has not been investigated because, as mentioned above, mammals have low levels of non-CG methylation, which is the type of DNA me...

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Section: Arabidopsis Telomeres Are Unmethylatedsupporting

confidence: 57%

“…Indeed, we found a strong positive correlation between the percentage of unconverted cytosines detected within 50TRs or mitochondrial reads and the percentage of unconverted reads using BS-Seeker2 ( Fig. 5B; Guo et al 2013).…”

Section: Arabidopsis Telomeres Are Unmethylatedmentioning

confidence: 75%

Novel features of telomere biology revealed by the absence of telomeric DNA methylation

et al. 2016

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“…Bisulfite-converted sequences were aligned to the C. reinhardtii nuclear genome (November 2011 assembly) and chloroplast genome using the BS-Seeker2 alignment pipeline version 2.0.5 (Guo et al, 2013). Default whole-genome bisulfite alignment parameters were chosen with the following exceptions: Bowtie2 was used as the aligner, and local alignments were enabled.…”

Section: Bisulfite-treated Dna Sequencing Analysismentioning

confidence: 99%

Dynamic changes in the transcriptome and methylome of Chlamydomonas reinhardtii throughout its life cycle

et al. 2015

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; 0000-0001-9355-9564 (M.P.).The green alga Chlamydomonas reinhardtii undergoes gametogenesis and mating upon nitrogen starvation. While the steps involved in its sexual reproductive cycle have been extensively characterized, the genome-wide transcriptional and epigenetic changes underlying different life cycle stages have yet to be fully described. Here, we performed transcriptome and methylome sequencing to quantify expression and DNA methylation from vegetative and gametic cells of each mating type and from zygotes. We identified 361 gametic genes with mating type-specific expression patterns and 627 genes that are specifically induced in zygotes; furthermore, these sex-related gene sets were enriched for secretory pathway and alga-specific genes. We also examined the C. reinhardtii nuclear methylation map with base-level resolution at different life cycle stages. Despite having low global levels of nuclear methylation, we detected 23 hypermethylated loci in gene-poor, repeat-rich regions. We observed mating type-specific differences in chloroplast DNA methylation levels in plus versus minus mating type gametes followed by chloroplast DNA hypermethylation in zygotes. Lastly, we examined the expression of candidate DNA methyltransferases and found three, DMT1a, DMT1b, and DMT4, that are differentially expressed during the life cycle and are candidate DNA methylases. The expression and methylation data we present provide insight into cell type-specific transcriptional and epigenetic programs during key stages of the C. reinhardtii life cycle.Chlamydomonas reinhardtii is a unicellular, biflagellate species of green alga found primarily in fresh water and soil (Harris et al., 2009). C. reinhardtii is an important reference organism for diverse eukaryotic cellular and metabolic processes, including photosynthetic biology (Rochaix, 2001), flagellar function and biogenesis (Silflow and Lefebvre, 2001), nutrient homeostasis (Grossman, 2000;Merchant et al., 2006;Glaesener et al., 2013), and sexual cycles (Goodenough et al., 2007). The nuclear and chloroplast genomes of C. reinhardtii have been fully sequenced, enabling genomic and epigenomic analyses (Maul et al., 2002;Merchant et al., 2007). The approximately 112-Mb haploid C. reinhardtii nuclear genome comprises 17 chromosomes. The circular chloroplast DNA (cpDNA) genome is 203 kb and present in 80 to 100 copies per cell that are organized into eight to 10 nucleoprotein complexes called nucleoids, which are distributed through the stroma.Like many unicellular eukaryotes, C. reinhardtii has a biphasic life cycle where haploid cells can reproduce vegetatively by mitotic division or, alternatively, undergo a sexual cycle. Vegetative cells can propagate indefinitely when provided with nutrients and light. Upon nitrogen starvation, however, cells stop dividing and differentiate into gametes whose mating type (plus or minus) is determined genetically by an approximately 300-kb mating type locus on chromosome 6, with two haplotypes, MT+ and MT2 (Umen, 2011; De Hoff et al.,...

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“…The BS-sequencing libraries were sequenced using the Illumina HiSequation 2000 platform to generate up to 100 cycles in paired ends. The reads were aligned to the reference genome (TAIR10) using BS Seeker 2 (Guo et al 2013). To profile genome-wide DNA methylation, the methylation level for each covered cytosine in the genome is estimated as #C/(#C + #T), where #C represents the number of methylated reads and #T corresponds to the number of unmethylated reads.…”

Section: Whole-genome Bisulfite Sequencingmentioning

confidence: 99%

An Rtf2 Domain-Containing Protein Influences Pre-mRNA Splicing and Is Essential for Embryonic Development in Arabidopsis thaliana

et al. 2015

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Alternative splicing is prevalent in plants, but little is known about its regulation in the context of developmental and signaling pathways. We describe here a new factor that influences pre-messengerRNA (mRNA) splicing and is essential for embryonic development in Arabidopsis thaliana. This factor was retrieved in a genetic screen that identified mutants impaired in expression of an alternatively spliced GFP reporter gene. In addition to the known spliceosomal component PRP8, the screen recovered Arabidopsis RTF2 (AtRTF2), a previously uncharacterized, evolutionarily conserved protein containing a replication termination factor 2 (Rtf2) domain. A homozygous null mutation in AtRTF2 is embryo lethal, indicating that AtRTF2 is an essential protein. Quantitative RT-PCR demonstrated that impaired expression of GFP in atrtf2 and prp8 mutants is due to inefficient splicing of the GFP pre-mRNA. A genome-wide analysis using RNA sequencing indicated that 13-16% of total introns are retained to a significant degree in atrtf2 mutants. Considering these results and previous suggestions that Rtf2 represents an ubiquitin-related domain, we discuss the possible role of AtRTF2 in ubiquitin-based regulation of pre-mRNA splicing.KEYWORDS alternative splicing; C2HC2 zinc finger; intron retention; Rtf2 domain; ubiquitin ligase I T is increasingly recognized that cotranscriptional and posttranscriptional gene regulation is comparable to transcriptional regulation in intricacy and importance. Pre-mRNA splicing is a cotranscriptional process and a major determinant of transcript abundance and complexity (Reddy et al. 2013). Constitutive splicing refers to the use of only one set of splice sites to generate a single mature mRNA. By contrast, alternative splicing occurs when variable splice sites are selected, leading to the generation of more than one processed RNA product from a single pre-messenger RNA (mRNA). An individual gene can thus potentially encode multiple proteins, leading to a substantial increase in proteomic diversity (Chen and Manley 2009;Syed et al. 2012;Reddy et al. 2013).Recent work has established that alternative splicing is common in plants, affecting 60% of intron-containing genes . Alternative splicing has important roles in plant growth, development, abiotic stress tolerance, circadian rhythms, and pathogen defense (Staiger and Brown 2013). The most common outcome of alternative splicing in plants is intron retention Lan et al. 2013), which occurs when an intron fails to be spliced out of the premRNA. Retained introns frequently contain premature termination codons (PTCs) that can channel the transcript into the nonsense-mediated decay (NMD) pathway. Intron retention provides a means for "transcriptome tuning" (Braunschweig et al. 2014) and contributes to the post-transcriptional regulation of gene expression by reducing levels of inappropriately expressed transcripts Ge and Porse 2013).Alternative splicing is subject to elaborate regulation that relies on general and specific trans-acting factors as ...

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BS-Seeker2: a versatile aligning pipeline for bisulfite sequencing data

Cited by 369 publications

References 19 publications

Novel features of telomere biology revealed by the absence of telomeric DNA methylation

Novel features of telomere biology revealed by the absence of telomeric DNA methylation

Dynamic changes in the transcriptome and methylome of Chlamydomonas reinhardtii throughout its life cycle

An Rtf2 Domain-Containing Protein Influences Pre-mRNA Splicing and Is Essential for Embryonic Development in Arabidopsis thaliana

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