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...d three consecutive fragments corresponding to the first, middle and Nucleic Acids Research, 2014, Vol. 42, No. 9 5731 last 40 bp of the probe and we mapped those to the reference genome using Bowtie =-=(29)-=-. We used a base quality score of 20 for each base of each simulated read and asked Bowtie to retain alignments with a sum of mismatch qualities less than or equal to 200, which retains alignments wit...

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... Because ETANDEM does not predict mononucleotide repeats (repeats with unit length equal to one), we downloaded from the UCSC server annotated repeats predicted by the TandemRepeats Finder (TRF) tool =-=(25)-=-, and we selected all the mononucleotide repeats from this dataset. We included the mononucleotide repeats in order to obtain the most comprehensive set of repeats irrespective of the NGS platform and...

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...ith theGS-FLXTitaniumXL+ kit (Roche), which produces single-end reads up to 600 bp. Bioinformatic analysis of sequence data Wealigned reads to the reference genome using bwa-swwith default parameters =-=(30)-=-.We implemented a custom script to perform local alignment and identify the 45-bp long flanking regions in the reads mapping next to targeted repeats, and extract the segment corresponding to the repe...

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...n tandem repeats and at general variation compared to the reference genome and within the family. Selection of tandem repeats We downloaded the last human reference genome (hg19) from the UCSC server =-=(24)-=-. To retrieve regions with tandem repeats in this reference, we ran the tool ETANDEM available in the EMBOSS package (18) with default parameters. ETANDEMcalculates a consensus sequence for a putative...

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...ats (38) had to be restricted to short microsatellites. However, sequencing longer reads is especially crucial to study tandem repeat variability because this variability increases with repeat length =-=(39,40)-=-. Our success in genotyping a larger number of coding and potential regulatory tandems thus is mainly due to the longer reads that were obtained by theGS-FLX+Roche technology (up to 700 bp) by which w...

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...non-functional (RI) groups. Such approach yields an estimation of the overall portion of polymorphic repeats at about 9%. This variability rate is 7.4 times higher than that observed for SNPs (1.25%) =-=(43)-=-, though it was expected to be up to 1000-fold higher (2). A potential explanation 5740 Nucleic Acids Research, 2014, Vol. 42, No. 9 might relate to the unclear definition of tandem repetitive sequenc...

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...of alleles of an apparent third allele with a lower read number than R2, or slippage associated with the sequencing protocol. Polymorphism in coding repeats was analyzed with the PROVEAN Protein tool =-=(31)-=-. PROVEAN calculates a delta alignment score based on the reference and variant versions of a protein query sequence with respect to sequence homologs collected from the non-redundant (NR) protein dat...

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...s are equally important sources of phenotypic variability and disease in higher eukaryotes, including plants, metazoans, mammals and humans (8,9). For example, an intriguing study byFondon and Garner =-=(10)-=- uncovered a strong correlation between variation in repeats located in two key regulatory genes (Alx4 and Runx2) and skeletal morphology in dogs, suggesting that repeat variation in these genes may a...

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...and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com Nucleic Acids Research, 2014, Vol. 42, No. 9 5729 gene =-=(6)-=-.Most of this research was focused on simple model organisms such as Saccharomyces cerevisiae (7). However, recent findings suggest that repeats are equally important sources of phenotypic variability...

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...categories for genes spanning annotated microRNAs, regulatory elements (transcription factor binding sites) annotated in the ORegAnno database (27) and CpG Islands annotated also in the UCSC database =-=(28)-=-. Repeats that did not span any of these functional elements were classified as intergenic. To select the set of targeted repeats for sequencing, we first set the maximal repeat length (total length o...

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...bination events generate alleles that differ in the number of repeated units (called ‘copy numbers’). Compared to other genomic loci, the mutation rates of tandem repeats are 10 to 10 000 fold higher =-=(2)-=-. Because of this instability and apparent lack of genetic information, most tandem repeats were thought to be devoid of direct biological function and termed ‘junk’ DNA (3). Tandem repeats did prove ...

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...e 10 to 10 000 fold higher (2). Because of this instability and apparent lack of genetic information, most tandem repeats were thought to be devoid of direct biological function and termed ‘junk’ DNA =-=(3)-=-. Tandem repeats did prove extremely useful as genetic markers in fine-scale genotyping and forensics. They also provide an added advantage to genome-wide linkage studies because of their higher diver...

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...in the adhesion properties of the cells, allowing tuning of biofilm formation (2). Similarly, variable tandem repeats in promoters allow fine-tuning and rapid divergence of downstream gene expression =-=(7,18,19)-=-. In the human genome, evidence suggests that some tandem repeat polymorphisms are under positive selection in certain parts of the world (20). Despite their prevalence in functional parts of genomes ...

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...in the adhesion properties of the cells, allowing tuning of biofilm formation (2). Similarly, variable tandem repeats in promoters allow fine-tuning and rapid divergence of downstream gene expression =-=(7,18,19)-=-. In the human genome, evidence suggests that some tandem repeat polymorphisms are under positive selection in certain parts of the world (20). Despite their prevalence in functional parts of genomes ...

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...ced at the PCR amplification step during the library preparation. PCR stutters due to slippage are expected to be most frequent for those repeats having a short repetitive unit and a high copy number =-=(32)-=-.Moreover, PCR stuttering usually results in a PCR product that is one or two copies shorter or longer than the actual repeat, and the PCR yield of the stutter product is typically much lower than tha...

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...ream (300 bp after the 3´ UTR). We also created separate categories for genes spanning annotated microRNAs, regulatory elements (transcription factor binding sites) annotated in the ORegAnno database =-=(27)-=- and CpG Islands annotated also in the UCSC database (28). Repeats that did not span any of these functional elements were classified as intergenic. To select the set of targeted repeats for sequencin...

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...amaternal allele with one repetitive unit althoughwe cannot exclude a two-unit expansion of the other maternal allele. However, since a one-unit change is more likely than a gain of two units at once =-=(44,45)-=-, the former option is preferred. When focusing on repeats located within the coding part that were sequenced in all seven individuals, we found 17 repeats for which at least one variation was detecte...

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... symptoms are directly correlated with repeat copy number in a particular gene, since allelic differences in tandem repeat copy numbers can influence allelic expression, e.g. in case of the ATRX gene =-=(14)-=-. In addition, tandem repeat variability in certain genes (e.g. Thymidylate Synthase gene) are associated with a poor prognosis in a number of cancers (15,16). However, despite the high number of gene...

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...a PCR product that is one or two copies shorter or longer than the actual repeat, and the PCR yield of the stutter product is typically much lower than that of the PCR product with the correct length =-=(33,34)-=-. Bearing this in mind, we corrected for those apparent heterozygous genotype calls in which the copy numbers differed by one or two copies, e.g. 11 and 10 copies, and for which the number of reads wa...

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...emain understudied. A precise mapping of all tandem repeats in the human genome is not fully achieved, and the relevant literature often contains conflicting results. For example, the lobSTR software =-=(21)-=- called 45 461 microsatellite loci from a trio of sequenced genomes, far less than the 376 685 microsatellites detected by another study using the same genome (22). This is mostly due to the technical...

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...se, please contact journals.permissions@oup.com Nucleic Acids Research, 2014, Vol. 42, No. 9 5729 gene (6).Most of this research was focused on simple model organisms such as Saccharomyces cerevisiae =-=(7)-=-. However, recent findings suggest that repeats are equally important sources of phenotypic variability and disease in higher eukaryotes, including plants, metazoans, mammals and humans (8,9). For exa...

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...cerevisiae (7). However, recent findings suggest that repeats are equally important sources of phenotypic variability and disease in higher eukaryotes, including plants, metazoans, mammals and humans =-=(8,9)-=-. For example, an intriguing study byFondon and Garner (10) uncovered a strong correlation between variation in repeats located in two key regulatory genes (Alx4 and Runx2) and skeletal morphology in ...

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...ss-induced proteins are particularly enriched in repeats, whereas genes encoding metabolic enzymes are depleted. Strikingly, this functional enrichment is evolutionary conserved from yeasts to humans =-=(2,8,17)-=-. As several reports documented, variable tandem repeats can provide functional diversity allowing rapid evolution of phenotypes. In S. cerevisiae, gradual changes in intragenic tandem repeats in the ...

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...cluding the identification of novel disease-related repeats. INTRODUCTION Repetitive DNA sequences make up a significant portion of all genomes.Almost half of the human genome is comprised of repeats =-=(1)-=-. A subset of repeated DNA is composed of tandem repeats, which are stretches of DNA that consist of tandemly repeated short sequence units (e.g. CAG) next to each other. The terms microsatellites and...

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...amaternal allele with one repetitive unit althoughwe cannot exclude a two-unit expansion of the other maternal allele. However, since a one-unit change is more likely than a gain of two units at once =-=(44,45)-=-, the former option is preferred. When focusing on repeats located within the coding part that were sequenced in all seven individuals, we found 17 repeats for which at least one variation was detecte...

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...elatively low-throughput NGS methods GS-FLX and SMRT (Roche). Although new analysis software packages specifically designed for genotyping tandem repeats from short reads have been recently published =-=(21,23)-=-, they are only able to genotype repeats with short unit length and low copy numbers from Illumina whole-genome sequencing libraries. Here, we set out to develop a strategy that permits a more accurat...

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...ths of 450 bp had been validated for efficient genotyping of a selection of five microsatellites in 10 individuals (41), or a much larger pool of tandem repeats in several different microbial species =-=(42)-=-. In our study, we enlarged the selection of tandem repeats by number (>10 000) and range of characteristics (both micro- and minisatellites, total repeat size up to 250 bp, low and high copy numbers ...

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...ts. For example, the lobSTR software (21) called 45 461 microsatellite loci from a trio of sequenced genomes, far less than the 376 685 microsatellites detected by another study using the same genome =-=(22)-=-. This is mostly due to the technical difficulties associated with sequencing repeats. Although improved methods are being introduced, the current standard next generation sequencing (NGS) techniques ...

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...(4). In certain cancers, the mutational spectrum of microsatellites appears to be tumor-type specific, thus opening new avenues for the use of microsatellites as genetic markers for disease diagnosis =-=(5)-=-. While many tandem repeats (also called ‘repeats’ further on) are present in gene deserts, the accumulation of whole genome sequencing data showed that repeats are also present in functional (coding ...

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...g and rapid divergence of downstream gene expression (7,18,19). In the human genome, evidence suggests that some tandem repeat polymorphisms are under positive selection in certain parts of the world =-=(20)-=-. Despite their prevalence in functional parts of genomes and their association with almost 20 human neurological diseases, and despite their usefulness as genetic markers, tandem repeats remain under...

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...nd/or Illumina instruments, was analyzed for 8342 repeats with the majority (94.5%)<20 bp in total length (35). Similarly, whole genome paired-end Illumina sequencing in 200 Drosophila inbred strains =-=(36)-=- or human gastric cancer cell lines and primary tissues (37), as well as the targeted capture MiSeq sequencing of thousands of selected short tandem repeats (38) had to be restricted to short microsat...

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...arkers in fine-scale genotyping and forensics. They also provide an added advantage to genome-wide linkage studies because of their higher diversity compared to single nucleotide polymorphisms (SNPs) =-=(4)-=-. In certain cancers, the mutational spectrum of microsatellites appears to be tumor-type specific, thus opening new avenues for the use of microsatellites as genetic markers for disease diagnosis (5)...

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...th the majority (94.5%)<20 bp in total length (35). Similarly, whole genome paired-end Illumina sequencing in 200 Drosophila inbred strains (36) or human gastric cancer cell lines and primary tissues =-=(37)-=-, as well as the targeted capture MiSeq sequencing of thousands of selected short tandem repeats (38) had to be restricted to short microsatellites. However, sequencing longer reads is especially cruc...

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...a PCR product that is one or two copies shorter or longer than the actual repeat, and the PCR yield of the stutter product is typically much lower than that of the PCR product with the correct length =-=(33,34)-=-. Bearing this in mind, we corrected for those apparent heterozygous genotype calls in which the copy numbers differed by one or two copies, e.g. 11 and 10 copies, and for which the number of reads wa...

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... >500 individuals of the 1000 Genomes Project exome sequencing pilot study, performed on 454 and/or Illumina instruments, was analyzed for 8342 repeats with the majority (94.5%)<20 bp in total length =-=(35)-=-. Similarly, whole genome paired-end Illumina sequencing in 200 Drosophila inbred strains (36) or human gastric cancer cell lines and primary tissues (37), as well as the targeted capture MiSeq sequen...

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...cerevisiae (7). However, recent findings suggest that repeats are equally important sources of phenotypic variability and disease in higher eukaryotes, including plants, metazoans, mammals and humans =-=(8,9)-=-. For example, an intriguing study byFondon and Garner (10) uncovered a strong correlation between variation in repeats located in two key regulatory genes (Alx4 and Runx2) and skeletal morphology in ...

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...ee additional sources: repeats used for genotyping and forensic applications (see: http://www.cstl.nist.gov/biotech/ strbase/), repeats related to diseases (8) and repeats studied by Matsumoto et al. =-=(26)-=-. We classified all these repeats based on their location relative to the catalog of annotated genes in the following categories: coding, intron, 5´ UTR, 3´ UTR, upstream (1 kb before the 5´ UTR) and ...

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...ic expression, e.g. in case of the ATRX gene (14). In addition, tandem repeat variability in certain genes (e.g. Thymidylate Synthase gene) are associated with a poor prognosis in a number of cancers =-=(15,16)-=-. However, despite the high number of genes potentially affected by tandem repeats, and despite the growing evidence of the functional role of variable repeats, most studies that report genetic variat...

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...ic expression, e.g. in case of the ATRX gene (14). In addition, tandem repeat variability in certain genes (e.g. Thymidylate Synthase gene) are associated with a poor prognosis in a number of cancers =-=(15,16)-=-. However, despite the high number of genes potentially affected by tandem repeats, and despite the growing evidence of the functional role of variable repeats, most studies that report genetic variat...

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...ncing in 200 Drosophila inbred strains (36) or human gastric cancer cell lines and primary tissues (37), as well as the targeted capture MiSeq sequencing of thousands of selected short tandem repeats =-=(38)-=- had to be restricted to short microsatellites. However, sequencing longer reads is especially crucial to study tandem repeat variability because this variability increases with repeat length (39,40)....

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...ats (38) had to be restricted to short microsatellites. However, sequencing longer reads is especially crucial to study tandem repeat variability because this variability increases with repeat length =-=(39,40)-=-. Our success in genotyping a larger number of coding and potential regulatory tandems thus is mainly due to the longer reads that were obtained by theGS-FLX+Roche technology (up to 700 bp) by which w...

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...epeat size up to 250 bp. In previous reports, the GS-FLX systemwith maximal read lengths of 450 bp had been validated for efficient genotyping of a selection of five microsatellites in 10 individuals =-=(41)-=-, or a much larger pool of tandem repeats in several different microbial species (42). In our study, we enlarged the selection of tandem repeats by number (>10 000) and range of characteristics (both ...