Single-Stranded DNA Library Preparation Preferentially Enriches Short Maternal DNA in Maternal Plasma J.S.L. Vong, J.C.H. Tsang, P. Jiang, W.-S. Lee, T.Y.

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Single-Stranded DNA Library Preparation Preferentially Enriches Short Maternal DNA in Maternal Plasma J.S.L. Vong, J.C.H. Tsang, P. Jiang, W.-S. Lee, T.Y. Leung, K.C.A. Chan, R.W.K. Chiu, Y.M.D. Lo May 2017 www.clinchem.org/content/63/5/1031.full © Copyright 2017 by the American Association for Clinical Chemistry

Introduction Background Aim Recent studies have suggested that single-stranded DNA (ssDNA) library preparation can enrich short DNA species from the plasma of healthy individuals, cancer patients, and transplant recipients. Fetal DNA molecules in the maternal plasma are shorter than maternal DNA molecules, ssDNA library preparation may potentially enrich fetal DNA and provide substantial improvement in non-invasive prenatal testing. Aim To examine the genomic characteristics of ssDNA and dsDNA libraries in the profiling of circulating DNA in first- and third-trimester maternal plasma samples. To compare the performance of ssDNA and dsDNA libraries in the detection of fetal aneuploidy in non-invasive prenatal testing

Question Why is fetal fraction enrichment important in non-invasive prenatal testing by cell-free fetal DNA analysis?

Materials & Methods Study population Euploid maternal plasma from male fetus singleton pregnancy Third-trimester (Pre-delivery) maternal plasma (n=4) First-trimester (13th week) maternal plasma (n=6) Trisomy 21 maternal plasma (n=2) Library preparation methods dsDNA: Double-stranded DNA library (KAPA HTP Library Preparation Kit for Illumina, KAPA Biosystems) TS-ssDNA: Template-switching based single-stranded DNA library (DNA SMART ChIP-Seq Kit, Clontech) LIG-ssDNA: Adaptor-ligation based single-stranded DNA library (Accel-NGS 1S Plus DNA Library Kit, SWIFT Biosciences)

Materials & Methods Parameters for evaluation Fetal fraction by fetal-specific SNPs and chrY fraction estimation Total fetal fraction Size-range specific fetal fraction Coefficients of variation of chromosomal representation Euploid-T21 pregnancy separation by Z score

Question What are the differences among the various library preparation methods in terms of their molecular principles?

Figure 2. (A) The DNA size profiles of ssDNA and dsDNA libraries of third-trimester maternal plasma dsDNA: Double-stranded DNA library TS-ssDNA: Template-switching based single-stranded DNA library LIG-ssDNA: Adaptor-ligation based single-stranded DNA library

Figure 2. (B), The short DNA fraction; (D), overall fetal fraction; (E,F), short DNA fraction in maternal/fetal-specific DNA of ssDNA and dsDNA libraries of third-trimester maternal plasma

Figure 2. (G), DNA size profiles of autosomal/chromosome Y and maternal/fetal DNA in maternal/fetal-specific DNA of ssDNA and dsDNA libraries of third-trimester maternal plasma

Figure 3. (B), Overall short DNA fraction; (C, D), Short DNA fraction in maternal/fetal DNA; (E), Overall fetal fraction of ssDNA and dsDNA libraries of first- trimester maternal plasma

Figure 3. (G), Size-specific fetal fraction of ssDNA and dsDNA libraries of first-trimester maternal plasma

Figure 3. (I), Separation of trisomy 21 samples by z scores in ssDNA and dsDNA libraries

Question What contributes to the preferential enrichment of short maternal DNA in ssDNA library preparation methods?

Conclusions ssDNA library preparation methods, irrespective of their chemistry, enrich short DNA species in maternal plasma ssDNA libraries preparation preferentially enriched short maternal DNA over short fetal DNA dsDNA library preparation provide better performance than ssDNA methods in trisomy 21 detection in count-based non-invasive prenatal testing by massively parallel sequencing.