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High-resolution characterization of sequence signatures due to non-random cleavage of cell-free DNA.

Chandrananda D, Thorne NP, Bahlo M - BMC Med Genomics (2015)

Bottom Line: Research that further characterizes circulating DNA could substantially increase its diagnostic value by allowing the application of more sophisticated bioinformatics tools that lead to an improved signal to noise ratio in the sequencing data.This sequence structure can be harnessed to improve bioinformatics algorithms, in particular for CNV and structural variant detection.Descriptive measures for cell-free DNA features developed here could also be used in biomarker analysis to monitor the changes that occur during different pathological conditions.

View Article: PubMed Central - PubMed

Affiliation: Bioinformatics Division, The Walter and Eliza Hall Institute of Medical Research, Parkville, Melbourne, VIC, 3052, Australia. chandrananda@wehi.edu.au.

ABSTRACT

Background: High-throughput sequencing of cell-free DNA fragments found in human plasma has been used to non-invasively detect fetal aneuploidy, monitor organ transplants and investigate tumor DNA. However, many biological properties of this extracellular genetic material remain unknown. Research that further characterizes circulating DNA could substantially increase its diagnostic value by allowing the application of more sophisticated bioinformatics tools that lead to an improved signal to noise ratio in the sequencing data.

Methods: In this study, we investigate various features of cell-free DNA in plasma using deep-sequencing data from two pregnant women (>70X, >50X) and compare them with matched cellular DNA. We utilize a descriptive approach to examine how the biological cleavage of cell-free DNA affects different sequence signatures such as fragment lengths, sequence motifs at fragment ends and the distribution of cleavage sites along the genome.

Results: We show that the size distributions of these cell-free DNA molecules are dependent on their autosomal and mitochondrial origin as well as the genomic location within chromosomes. DNA mapping to particular microsatellites and alpha repeat elements display unique size signatures. We show how cell-free fragments occur in clusters along the genome, localizing to nucleosomal arrays and are preferentially cleaved at linker regions by correlating the mapping locations of these fragments with ENCODE annotation of chromatin organization. Our work further demonstrates that cell-free autosomal DNA cleavage is sequence dependent. The region spanning up to 10 positions on either side of the DNA cleavage site show a consistent pattern of preference for specific nucleotides. This sequence motif is present in cleavage sites localized to nucleosomal cores and linker regions but is absent in nucleosome-free mitochondrial DNA.

Conclusions: These background signals in cell-free DNA sequencing data stem from the non-random biological cleavage of these fragments. This sequence structure can be harnessed to improve bioinformatics algorithms, in particular for CNV and structural variant detection. Descriptive measures for cell-free DNA features developed here could also be used in biomarker analysis to monitor the changes that occur during different pathological conditions.

No MeSH data available.


Related in: MedlinePlus

Autosomal fragment lengths originating at regions annotated for alpha repeat elements and two micro-satellite types. The number of fragments used to calculate the size distribution is depicted in the legend beside each repeat category. The repeat specific profiles are superimposed over the genome-wide profile in sample I1_M_plasma for comparison purposes
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Fig4: Autosomal fragment lengths originating at regions annotated for alpha repeat elements and two micro-satellite types. The number of fragments used to calculate the size distribution is depicted in the legend beside each repeat category. The repeat specific profiles are superimposed over the genome-wide profile in sample I1_M_plasma for comparison purposes

Mentions: We make use of the Repbase database and RepeatMasker annotation for this analysis [57]. When comparing 32 broad categories of abundant repeats as per the class/family classification in RepeatMasker, there appears to be no difference in the fragment sizes with the density curves overlaying each other closely (Additional file 5). Narrowing the scope to 50 more specific repeat types within the broader classifications also shows no gross imbalances in fragment lengths except in three categories (Fig. 4). Details for the 50 repeat types analyzed are provided in Additional file 6.Fig. 4


High-resolution characterization of sequence signatures due to non-random cleavage of cell-free DNA.

Chandrananda D, Thorne NP, Bahlo M - BMC Med Genomics (2015)

Autosomal fragment lengths originating at regions annotated for alpha repeat elements and two micro-satellite types. The number of fragments used to calculate the size distribution is depicted in the legend beside each repeat category. The repeat specific profiles are superimposed over the genome-wide profile in sample I1_M_plasma for comparison purposes
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4469119&req=5

Fig4: Autosomal fragment lengths originating at regions annotated for alpha repeat elements and two micro-satellite types. The number of fragments used to calculate the size distribution is depicted in the legend beside each repeat category. The repeat specific profiles are superimposed over the genome-wide profile in sample I1_M_plasma for comparison purposes
Mentions: We make use of the Repbase database and RepeatMasker annotation for this analysis [57]. When comparing 32 broad categories of abundant repeats as per the class/family classification in RepeatMasker, there appears to be no difference in the fragment sizes with the density curves overlaying each other closely (Additional file 5). Narrowing the scope to 50 more specific repeat types within the broader classifications also shows no gross imbalances in fragment lengths except in three categories (Fig. 4). Details for the 50 repeat types analyzed are provided in Additional file 6.Fig. 4

Bottom Line: Research that further characterizes circulating DNA could substantially increase its diagnostic value by allowing the application of more sophisticated bioinformatics tools that lead to an improved signal to noise ratio in the sequencing data.This sequence structure can be harnessed to improve bioinformatics algorithms, in particular for CNV and structural variant detection.Descriptive measures for cell-free DNA features developed here could also be used in biomarker analysis to monitor the changes that occur during different pathological conditions.

View Article: PubMed Central - PubMed

Affiliation: Bioinformatics Division, The Walter and Eliza Hall Institute of Medical Research, Parkville, Melbourne, VIC, 3052, Australia. chandrananda@wehi.edu.au.

ABSTRACT

Background: High-throughput sequencing of cell-free DNA fragments found in human plasma has been used to non-invasively detect fetal aneuploidy, monitor organ transplants and investigate tumor DNA. However, many biological properties of this extracellular genetic material remain unknown. Research that further characterizes circulating DNA could substantially increase its diagnostic value by allowing the application of more sophisticated bioinformatics tools that lead to an improved signal to noise ratio in the sequencing data.

Methods: In this study, we investigate various features of cell-free DNA in plasma using deep-sequencing data from two pregnant women (>70X, >50X) and compare them with matched cellular DNA. We utilize a descriptive approach to examine how the biological cleavage of cell-free DNA affects different sequence signatures such as fragment lengths, sequence motifs at fragment ends and the distribution of cleavage sites along the genome.

Results: We show that the size distributions of these cell-free DNA molecules are dependent on their autosomal and mitochondrial origin as well as the genomic location within chromosomes. DNA mapping to particular microsatellites and alpha repeat elements display unique size signatures. We show how cell-free fragments occur in clusters along the genome, localizing to nucleosomal arrays and are preferentially cleaved at linker regions by correlating the mapping locations of these fragments with ENCODE annotation of chromatin organization. Our work further demonstrates that cell-free autosomal DNA cleavage is sequence dependent. The region spanning up to 10 positions on either side of the DNA cleavage site show a consistent pattern of preference for specific nucleotides. This sequence motif is present in cleavage sites localized to nucleosomal cores and linker regions but is absent in nucleosome-free mitochondrial DNA.

Conclusions: These background signals in cell-free DNA sequencing data stem from the non-random biological cleavage of these fragments. This sequence structure can be harnessed to improve bioinformatics algorithms, in particular for CNV and structural variant detection. Descriptive measures for cell-free DNA features developed here could also be used in biomarker analysis to monitor the changes that occur during different pathological conditions.

No MeSH data available.


Related in: MedlinePlus