scholarly journals Single-stranded DNA library preparation uncovers the origin and diversity of ultrashort cell-free DNA in plasma

2016 ◽  
Vol 6 (1) ◽  
Author(s):  
Philip Burnham ◽  
Min Seong Kim ◽  
Sean Agbor-Enoh ◽  
Helen Luikart ◽  
Hannah A. Valantine ◽  
...  
Keyword(s):  
Library Preparation ◽  
Cell Free Dna ◽  
Single Stranded Dna ◽  
Dna Library ◽  
Free Dna

scholarly journals Single-stranded DNA library preparation uncovers the origin and diversity of ultrashort cell-free DNA in plasma

2015 ◽  
Author(s):  
Philip Burnham ◽  
Min Seong Kim ◽  
Sean Agbor-Enoh ◽  
Helen Luikart ◽  
Hannah A Valantine ◽  
...  
Keyword(s):  
Lung Transplant ◽  
Preparation Method ◽  
Fragmentation Pattern ◽  
Library Preparation ◽  
Transplant Recipients ◽  
Cell Free Dna ◽  
Single Stranded Dna ◽  
Free Dna ◽  
Lung Transplant Recipients ◽  
Library Preparation Method

Circulating cell-free DNA (cfDNA) is emerging as a powerful monitoring tool in cancer, pregnancy and organ transplantation. Nucleosomal DNA, the predominant form of cfDNA in blood, can be readily adapted for sequencing via ligation of double-stranded DNA (dsDNA) adapters. dsDNA library preparation, however, is insensitive to ultrashort, degraded and single-stranded cfDNA. Drawing inspiration from recent technical advances in ancient genome analyses, we have applied a single-stranded DNA (ssDNA) library preparation method to sequencing of cfDNA in the plasma of lung transplant recipients (40 samples, six patients). We found that the ssDNA library preparation yields a greater portion of sub-100 bp DNA, as well as an increased relative abundance of human mitochondrial cfDNA (10.7x) and microbial cfDNA (71.3x). We report the fragmentation pattern of mitochondrial, nuclear genomic and microbial cfDNA over a broad fragment length range. We furthermore report the first observation of donor-specific mitochondrial cfDNA in the circulation of lung transplant recipients. We found that donor-specific mitochondrial cfDNA molecules are significantly shorter than those specific to the recipient. The higher yield of viral, microbial and fungal sequences that result from the single-stranded ligation approach reduces the cost and increase the sensitivity of cfDNA-based monitoring for infectious complications after transplantation. An ssDNA library preparation method provides a more informative window into understudied forms of cfDNA, including mitochondrial and microbial derived cfDNA and short fragment nuclear genomic cfDNA, while retaining information provided by standard dsDNA library preparation methods.

Single-stranded DNA library preparation for the sequencing of ancient or damaged DNA

2013 ◽  
Vol 8 (4) ◽  
pp. 737-748 ◽  
Author(s):  
Marie-Theres Gansauge ◽  
Matthias Meyer
Keyword(s):  
Library Preparation ◽  
Single Stranded Dna ◽  
Dna Library ◽  
Damaged Dna

scholarly journals SALP, a new single-stranded DNA library preparation method especially useful for the high-throughput characterization of chromatin openness states

2017 ◽  
Author(s):  
Jian Wu ◽  
Wei Dai ◽  
Ling Wu ◽  
Jinke Wang
Keyword(s):  
Biological Sciences ◽  
High Throughput ◽  
High Sensitivity ◽  
Single Strand ◽  
Library Preparation ◽  
Single Stranded Dna ◽  
Dna Library ◽  
Next Generation Sequencing Ngs ◽  
Multiple Cells

AbstractBased on a novel kind of single strand adaptor (SSA), this study developed a new method to construct next-generation sequencing (NGS) library, named as SALP, representing Single strand Adaptor Library Preparation. The key creativity of the method lies in the design and verification of a special adaptor that can be efficiently linked to the 3′ end of single-stranded DNA, which is a double-stranded oligonucleotide with a 3′ overhang of 3 random nucleotides. This method can start with the denatured DNAs or chromatins fragmented by different methods such as Tn5 tagmentation, enzyme digestion and sonication. When applied to Tn5-tagmented chromatin, SALP overcomes the key limitation of the current ATAC-seq method and develops a high-throughput NGS library construction and sequencing approach, SALP-seq, which can be used to comparatively characterize the chromatin openness state of multiple cells simply and unbiasly. In this way, the comparative chromatin openness states of four different cell lines, including GM12878, HepG2, HeLa and 293T, were successfully characterized. This study also demonstrated that SALP-seq could characterize the chromatin openness states with 105 to 500 cells, indicating the high sensitivity of SALP-seq in characterizing chromatin state of cells. SALP should have wide applications in the future biological sciences and biomedicine.

scholarly journals Advantages of Single-Stranded DNA Over Double-Stranded DNA Library Preparation for Capturing Cell-Free Tumor DNA in Plasma

2019 ◽  
Vol 24 (1) ◽  
pp. 95-101 ◽  
Author(s):  
Jing Zhu ◽  
Jinyong Huang ◽  
Peng Zhang ◽  
Qianxia Li ◽  
Manish Kohli ◽  
...  
Keyword(s):  
Library Preparation ◽  
Single Stranded Dna ◽  
Dna Library ◽  
Double Stranded Dna ◽  
Tumor Dna

scholarly journals Short single-stranded DNA with putative non-canonical structures comprises a novel class of plasma cell-free DNA

2021 ◽  
Author(s):  
Osamu Hisano ◽  
Takashi Ito ◽  
Fumihito Miura
Keyword(s):  
Binding Sites ◽  
Open Chromatin ◽  
Cell Free Dna ◽  
Single Stranded Dna ◽  
Dna Structures ◽  
Free Dna ◽  
G Quadruplex ◽  
Labeling Method ◽  
Purification Protocol ◽  
Library Preparation Protocol

Cell-free DNA (cfDNA) in human blood is currently investigated as a minimally invasive, highly informative biomarker. Here, we aimed to investigate the existence of the shorter cfDNA fragments in the blood. Using an improved cfDNA purification protocol and a 3′-end-labeling method, we found DNA fragments of approximately 50 nucleotides in human plasma, present at a molar concentration comparable to that of the nucleosome-sized fragments. These short fragments cannot be recovered by widely used cfDNA isolation methods, and are composed of single-stranded DNA (ssDNA), thus escaping detection in previous studies. We established a library-preparation protocol based on our unique ssDNA ligation technique and applied it to the isolated cfDNA. Deep sequencing of these libraries revealed that the short fragments are derived from hundreds of thousands of genomic sites in open chromatin regions and enriched with transcription factor-binding sites. Remarkably, antisense strands of putative G-quadruplex motifs occupy as much as one-third of peaks called with these short fragments. Hence, we propose a novel class of plasma cfDNA composed of short single-stranded fragments that potentially form non-canonical DNA structures.

scholarly journals Single-stranded DNA library preparation from highly degraded DNA usingT4DNA ligase

2017 ◽  
pp. gkx033 ◽  
Author(s):  
Marie-Theres Gansauge ◽  
Tobias Gerber ◽  
Isabelle Glocke ◽  
Petra Korlević ◽  
Laurin Lippik ◽  
...  
Keyword(s):  
Degraded Dna ◽  
Library Preparation ◽  
Single Stranded Dna ◽  
Dna Library

scholarly journals Single-Stranded DNA Library Preparation Does Not Preferentially Enrich Circulating Tumor DNA

2017 ◽  
Vol 63 (10) ◽  
pp. 1656-1659 ◽  
Author(s):  
Tina Moser ◽  
Peter Ulz ◽  
Qing Zhou ◽  
Samantha Perakis ◽  
Jochen B Geigl ◽  
...  
Keyword(s):  
Circulating Tumor Dna ◽  
Library Preparation ◽  
Single Stranded Dna ◽  
Dna Library ◽  
Tumor Dna

scholarly journals A ligation-based single-stranded library preparation method to analyze cell-free DNA and synthetic oligos

BMC Genomics ◽  
2019 ◽  
Vol 20 (1) ◽  
Author(s):  
Christopher J. Troll ◽  
Joshua Kapp ◽  
Varsha Rao ◽  
Kelly M. Harkins ◽  
Charles Cole ◽  
...  
Keyword(s):  
Preparation Method ◽  
Organ Transplant ◽  
Genomic Analysis ◽  
Library Preparation ◽  
Cell Free Dna ◽  
Preparation Methods ◽  
Prenatal Health ◽  
Free Dna ◽  
Fragmented Dna ◽  
Library Preparation Method

Abstract Background Cell-free DNA (cfDNA), present in circulating blood plasma, contains information about prenatal health, organ transplant reception, and cancer presence and progression. Originally developed for the genomic analysis of highly degraded ancient DNA, single-stranded DNA (ssDNA) library preparation methods are gaining popularity in the field of cfDNA analysis due to their efficiency and ability to convert short, fragmented DNA into sequencing libraries without altering DNA ends. However, current ssDNA methods are costly and time-consuming. Results Here we present an efficient ligation-based single-stranded library preparation method that is engineered to produce complex libraries in under 2.5 h from as little as 1 nanogram of input DNA without alteration to the native ends of template molecules. Our method, called Single Reaction Single-stranded LibrarY or SRSLY, ligates uniquely designed Next-Generation Sequencing (NGS) adapters in a one-step combined phosphorylation/ligation reaction that foregoes end-polishing. Using synthetic DNA oligos and cfDNA, we demonstrate the efficiency and utility of this approach and compare with existing double-stranded and single-stranded approaches for library generation. Finally, we demonstrate that cfDNA NGS data generated from SRSLY can be used to analyze DNA fragmentation patterns to deduce nucleosome positioning and transcription factor binding. Conclusions SRSLY is a versatile tool for converting short and fragmented DNA molecules, like cfDNA fragments, into sequencing libraries while retaining native lengths and ends.

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