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 ◽  
...  

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.

2021 ◽  
Vol 40 (4) ◽  
pp. S320-S321
Author(s):  
J.B. Smith ◽  
J. Stumph ◽  
Y. Bryan ◽  
R. Peterson ◽  
M.P. Steele ◽  
...  

2021 ◽  
Vol 40 (4) ◽  
pp. S338-S339
Author(s):  
M.B. Keller ◽  
C. Mutebi ◽  
P. Shah ◽  
D. Levine ◽  
S. Aryal ◽  
...  

2020 ◽  
Vol 9 (7) ◽  
pp. 2307
Author(s):  
Yan Y. Sanders

The main challenge for a positive long-term outcome in lung transplantation is the lack of early detection for chronic lung allograft dysfunction (CLAD). With advancements in technology, an increasing number of studies demonstrate that cell-free DNA (cfDNA) in body fluids could be used as a marker for disease diagnosis, prognosis or monitoring response to treatment. A previous report from this journal found the joint assessment of cfDNA and CXCL10 from brochoalveolar lavage (BAL) could determine the subphenotypes of CLAD and predict lung transplant survival. This is an exciting attempt in monitoring the progress for lung transplant recipients. More studies and better understanding of cfDNA are needed to develop an accessible and reliable biomarker to monitor the progress of CLAD to improve the long-term survival for lung transplant recipients.


2015 ◽  
Vol 76 ◽  
pp. 68 ◽  
Author(s):  
Jun Zou ◽  
Brian Duffy ◽  
Nancy Steward ◽  
Ramsey Hachem ◽  
Thalachallour Mohanakumar

BMC Genomics ◽  
2019 ◽  
Vol 20 (1) ◽  
Author(s):  
Christopher J. Troll ◽  
Joshua Kapp ◽  
Varsha Rao ◽  
Kelly M. Harkins ◽  
Charles Cole ◽  
...  

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.


2017 ◽  
Vol 78 (4) ◽  
pp. 342-349 ◽  
Author(s):  
Jun Zou ◽  
Brian Duffy ◽  
Michael Slade ◽  
Andrew Lee Young ◽  
Nancy Steward ◽  
...  

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