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Using TGIRT for ssDNA-seq of cell free DNA in human plasma
- 1. Douglas C. Wu @wckdouglas wckdouglas.github.io Lambowitz’s Lab CBRS Open House 04/18/2017 Use of Thermostable Group II Intron Reverse Transcriptase (TGIRT) for Single-Stranded DNA-seq of Cell-Free DNA in Human Plasma
- 2. Human plasma cell-free DNA provides epigenetic information Snyder and Kricher et al. (2016) Cell
- 3. TGIRT-seq is a Streamlined Library Prep for ssDNA-seq Snyder and Kricher et al. (2016) Cell Wu and Lambowitz (In preparation) http://www.entechus.com/hubfs/images/Server_Rack_Comparison.jpg?t=1489167725226 https://assets.illumina.com/content/dam/illumina-marketing/images/systems/v2/systems-carousel/system-carousel-nextseq500-left.png 20 min 20 min 60 min ~2 ng (3) Template-switching by TGIRT Alkaline treatment cDNA clean-up (4) Adaptor ligation by thermostable 5’ AppDNA/RNA ligase R2 RNA 3’-Blocker 5’ 5’ 3’-N R2R DNA 5’ 3’OH TGIRT cDNA clean-up (5) PCR amplification 5’-App3’-Blocker 5’3’ R1R R2R 5’ R2R R2P5 3’ 5’ Barcode+P7R1 ’ 5’ 5’P DNA nick P (2) Dephosphorylation& denaturation 5’ 3’ OH 5’3’ OH (1) Plasma DNA Target DNA (-) 5’ 5’ 5’ 3’ OH 3’ OH 3’ OH5’ 3’ OH 5’ 3’ OH Target DNA (-) Target DNA (+) Target DNA (+) Target DNA (-) UMI UMI 3’ R1R Supplemental Figures GSAF RCTF
- 4. 167 nt 0.0 0.5 1.0 1.5 2.0 2.5 0 50 100 150 200 250 300 350 400 Fragment length (nt) Percentreads ssDNA−seq TGIRT−seq Long(120−180nt)Short(35−80nt) −1000 −800 −600 −400 −200 0 200 400 600 800 1000 −1 0 1 2 0 5 Position relative to CTCF binding sites (bp) ScaledWPS TGIRT−seq TA/TT /CG/CC b Wu and Lambowitz (In preparation) TGIRT-seq identifies tissue-specific nucleosome positionings in healthy donor
- 5. Neutrophils Lymphocytes Neutrophils: 58% Lymphocytes: 16% Lungs: 8.6% Adipose tissues: 6.3% Adrenal glands: 5.6% Liver: 2.6% Heart: 1.4% Brain: 1.2% Pancreas: 0.25% Small intestines: 1.8e−16% Colon: 3.1e−17% Esophagus: −2.6e−15% b 0M 50M 100M 150M 200M 0M 50M 100M150M 200M 0M 50M 100M 150M 0M 50M 100M 150M 0M 50M 100M 150M 0M 50M 100M150M 0M 50M 100M 150M 0M 50M 100M 0M 50M 100M 0M 50M 100M 0M 50M 100M 0M 50M 100M 0M 50M 100M 0M 50M 100M 0M 50M 100M 0M 50M 0M 50M 0M 50M 0M 50M 0M 50M 0M 0M 50M chr1 chr2 chr3 chr4chr5 chr6 chr7 chr8chr9 chr10 chr11 chr12 chr13 chr14chr15chr16 chr17 chr18 chr19 chr20chr21chr22 Neutrophils Lymphocytes Lungs Adipose tissues Adrenal glands Liver Heart Brain Pancreas Small intestines Colon Esophagus TGIRT-seq on bisulfite-treated cfDNA identifies tissue-specific 5mC in healthy donor Wu and Lambowitz (In preparation)
- 6. Conclusion •GSAF is great 💯 •TGIRTs enable efficient ssDNA-seq for analysis of cfDNA in human plasma and other bodily fluids •Identification of protein binding features of cfDNA provides information about the tissue-of-origin and has potential diagnostic applications •TGIRT DNA-seq should also be applicable to other damaged DNA samples: • Ancient DNA • FFPE DNA • ChIP-seq •DNA/RNA co-sequencing: metagenomics?
- 7. Lambowitz Lab Sabine Mohr, Ph.D (Yidan Qin, Ph.D.) Ryan Nottingham, Ph.D. Jun Yao, Ph.D. and other lab members. GSAF Michael Wilson, Ph. D Jessica Podnar Gabriella Huerta and others. Disclosure: Thermostable group II intron RTs and methods for their use are subject to issued patents and patent applications that are licensed by the University of Texas at Austin to InGex, LLC, which sell TGIRT enzymes and kits and sublicenses to others for commercial development. A.M.L., some lab members, and UT Austin are minority equity holders in InGex, LLC, and receive royalty payments from commercial use of the technologies. RCTF Anna Battenhouse Eric J Rostetter and others. Support NIH GM37949 and GM37951 Welch Foundation