Journal: bioRxiv

Article Title: DNA damage is a major cause of sequencing errors, directly confounding variant identification

doi: 10.1101/070334

Figure Lengend Snippet: A. Principle of the GIV score : Illumina adaptors are directional in nature, enabling consistent paired end sequencing within clusters. This property results in sequencing of the original strand orientation in the R1 reads (from the P5 adaptor) whereas the reverse complement orientation is read in the R2 reads (from the P7 adaptor). As damage affects only one base of a pair, damage such as 8-oxo-dG leads to an excess of G to T transversion errors when R1 is mapped to a reference genome, whereas, the R2 reads will show an excess of the reverse complement of G to T, i.e. C to A transversion errors, instead. As a consequence, there is a global imbalance in the number of G to T variants in R1 compared to R2 sequences. This imbalance is specific to damage and is the basis of the GIV score (see ). B. Overall fraction of G to T variants (normalized to the total number of G) for R1 and the reverse complement of R2 sequences. Different buffers were used during acoustic shearing (x-axis). Data in red were from samples that were not repaired and data in blue were from samples that were repaired. Each point corresponds to a random sampling of 2 million sequence positions. All samples are derived from the same human genomic DNA. C. Variant profile: The fraction of G to T and C to A variants in R1 and R2 sequences are plotted as a function of the read (R1 or R2) and the positions on the read (in bp). D. Correlation (R=0.97) between the degree of damage that is repaired by the DNA repair enzyme cocktail and GIV for G to T variant (GIV G_T ) and T to A variant (GIV T_A ).

Article Snippet: To introduce oxidative damage, purified human liver genomic DNA samples (BioChain Institute, Inc.) were fragmented by shearing in 1x TE (10 mM Tris pH 8, 1 mM EDTA), 0.1x TE (1 mM Tris pH 8, 0.1 mM EDTA) or water using a Covaris S2 to produce 200 bp average fragment size as determined by Bioanalyzer (Agilent Technologies) analysis.

Techniques: Sequencing, Sampling, Derivative Assay, Variant Assay

Journal: bioRxiv

Article Title: DNA damage is a major cause of sequencing errors, directly confounding variant identification

doi: 10.1101/070334

Figure Lengend Snippet: Genomic DNA was sheared in 0.1xTE buffer and subjected to library preparation and cancer panel target enrichment. DNA repair treatment was performed postshearing and prior to library preparation. The control represents untreated DNA. A. Overall G to T variant profiles across reads R1 and R2 with (red) and without (blue) repair treatment. B. Variant spectrum at a frequency less than 1% (left panel), 1 to 5% (center left panel), 6 to 10% (center right panel) and more than 10% (right panel). C. Same as for B except that only R1 reads were used for variant calling.

Article Snippet: To introduce oxidative damage, purified human liver genomic DNA samples (BioChain Institute, Inc.) were fragmented by shearing in 1x TE (10 mM Tris pH 8, 1 mM EDTA), 0.1x TE (1 mM Tris pH 8, 0.1 mM EDTA) or water using a Covaris S2 to produce 200 bp average fragment size as determined by Bioanalyzer (Agilent Technologies) analysis.

Techniques: Variant Assay

Journal:

Article Title: Localization of retinaldehyde dehydrogenases and retinoid binding proteins to sustentacular cells, glia, Bowman's gland cells and stroma: potential sites of retinoic acid synthesis in the postnatal rat olfactory organ

doi: 10.1002/cne.20904

Figure Lengend Snippet: PCR reactions with primers specific for RALDH 1, 2, and 3 and ALDH-PB were carried out as described in Materials and Methods using either 100 ng postnatal rat olfactory cDNA (A) or 100 ng genomic DNA (B). A. mRNAs encoding RALDH 1, 2, and 3 are expressed in postnatal rat olfactory tissue. Shown is an image of the gel-separated, RT-PCR products from reactions containing cDNA prepared from DNAse-I treated olfactory tissue RNA. Bands were visualized with SYBR Green I. Lane 1, 100 bp ladder; lane 2, RALDH 1; lane 3, ALDH-PB; lane 4, RALDH 2; lane 5, RALDH 3; lane 6, minus cDNA control reaction containing primers for all ALDH-PB / RALDH transcripts. B. ALDH-PB mRNA was not detected in olfactory tissue, but the ALDH-PB primers amplify authentic gene products from rat genomic DNA. Shown is an image of gel-separated, PCR products from reactions containing rat genomic DNA. Bands were visualized with SYBR Green I. Lane 1, RALDH 1; lane 2, ALDH-PB. Markers designating migration points of 300 and 400 bp ladder transcripts that were run on the gel are shown to the left of the image.

Article Snippet: PCR reactions were also prepared from 100 ng rat genomic DNA (rat liver genomic DNA #D4434149, BioChain Institute, Hayward, CA) using primer sets for RALDH 1 and ALDH-PB.

Techniques: Reverse Transcription Polymerase Chain Reaction, SYBR Green Assay, Control, Migration