bisulfite sequencing analysis  (Zymo Research)

Journal: Bioinformatics

Article Title: ABSP: an automated R tool to efficiently analyze region-specific CpG methylation from bisulfite sequencing PCR

doi: 10.1093/bioinformatics/btad008

Figure Lengend Snippet: Analysis strategy differences between the two bisulfite sequencing PCR approaches, direct-BSP and cloning-BSP. As the direct-BSP method consists in sequencing the mix of PCR products, the methylation calculation based on chromatogram C/T peak heights gives directly an estimation of the CpG methylation level that can be replicated for statistical significance determination. In the cloning-BSP method, as the CpG methylation status in each clone is revealed by the chromatogram peak heights as well, the sequencing of several clones gives an estimation of the CpG methylation proportion

Article Snippet: However, ESME was developed in the early stages of the BSP technology, and nowadays basecallers have been improved and do not exaggerate the missing base, its normalization step is therefore no longer required and may introduce biases in methylation percentages calculated (Methylation Analysis by Bisulfite Sequencing: Chemistry, Products and Protocols from Applied Biosystems, 2007, https://assets.thermofisher.com/TFS-Assets/LSG/manuals/cms_039258.pdf ).

Techniques: Methylation Sequencing, Cloning, Sequencing, Methylation, CpG Methylation Assay, Clone Assay

Journal: Bioinformatics

Article Title: ABSP: an automated R tool to efficiently analyze region-specific CpG methylation from bisulfite sequencing PCR

doi: 10.1093/bioinformatics/btad008

Figure Lengend Snippet: General workflow of ABSP. The analysis is divided into two main steps: the individual analysis and grouped analysis . The first one serves to control the sequencing quality and compute methylation levels for each individual sample, whereas the second one gathers all sample methylation results to generate visualization plots and process comparative statistics between groups

Article Snippet: However, ESME was developed in the early stages of the BSP technology, and nowadays basecallers have been improved and do not exaggerate the missing base, its normalization step is therefore no longer required and may introduce biases in methylation percentages calculated (Methylation Analysis by Bisulfite Sequencing: Chemistry, Products and Protocols from Applied Biosystems, 2007, https://assets.thermofisher.com/TFS-Assets/LSG/manuals/cms_039258.pdf ).

Techniques: Control, Sequencing, Methylation

Journal: Bioinformatics

Article Title: ABSP: an automated R tool to efficiently analyze region-specific CpG methylation from bisulfite sequencing PCR

doi: 10.1093/bioinformatics/btad008

Figure Lengend Snippet: Output results from ABSP analysis. ( A ) Portions of the trimmed chromatogram traces from forward and reverse sequencing reads of the CDH1 sequence in the high-methylated DNA sample, replicate #3. The CG #8 is highlighted and corresponds to the eighth CG dinucleotide of the reference sequence. ( B ) Table of quality control summary after alignments of trimmed sequencing results with the reference sequence. ( C ) Tables of the methylation calculation per CpG using peak height values from both chromatogram traces. ( D ) Table of combined methylation data from both sequencing results, with the methylation average and standard deviation per CpG. ( E ) Lollipop-style plot of CpG methylation levels on the CDH1 sequence, with all the samples displayed and CpG sites placed proportionally to their coordinates. The CG #8 of the high-methylated DNA #3 sample, detailed in the individual analysis panel, is highlighted by encircling targeted CG in the lollipop-style plot and corresponds to the fourth CpG site on the covered sequence. The crosses represent unavailable data for CpG sites not covered by the trimmed sequencing results in some samples. ( F ) Methylation profile plot displaying average methylation levels of CpG along the CDH1 sequence in each group as a line plot. Symbols represent significance levels of Kruskal–Wallis test P -values (* P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.0001). ( G ) Lollipop-style plot of average CpG methylation levels on the CDH1 sequence in each group. ( H ) Boxplot of the CpG #4 average methylation levels in each group. ( I ) Boxplot of the methylation percentage means of all CpG positions on the CDH1 sequence in each group. In boxplots, symbols represent significance levels of Student’s t -test P -values (* P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.0001)

Article Snippet: However, ESME was developed in the early stages of the BSP technology, and nowadays basecallers have been improved and do not exaggerate the missing base, its normalization step is therefore no longer required and may introduce biases in methylation percentages calculated (Methylation Analysis by Bisulfite Sequencing: Chemistry, Products and Protocols from Applied Biosystems, 2007, https://assets.thermofisher.com/TFS-Assets/LSG/manuals/cms_039258.pdf ).

Techniques: Sequencing, Methylation, Control, Standard Deviation, CpG Methylation Assay