Sequencing artifacts derived from a library preparation method using enzymatic fragmentation

Abstract

DNA fragmentation is a fundamental step during library preparation in hybridization capturebased, short-read sequencing. Ultra-sonication has been used thus far to prepare DNA of an appropriate size, but this method is associated with a considerable loss of DNA sample. More recently, studies have employed library preparation methods that rely on enzymatic fragmentation with DNA endonucleases to minimize DNA loss, particularly in nano-quantity samples. Yet, despite their wide use, the effect of enzymatic fragmentation on the resultant sequences has not been carefully assessed. Here, we used pairwise comparisons of somatic variants of the same tumor DNA samples prepared using ultrasonic and enzymatic fragmentation methods. Our analysis revealed a substantially larger number of recurrent artifactual SNVs/indels in endonuclease-treated libraries as compared with those created through ultrasonication. These artifacts were marked by palindromic structure in the genomic context, positional bias in sequenced reads, and multi-nucleotide substitutions. Taking advantage of these distinctive features, we developed a filtering algorithm to distinguish genuine somatic mutations from artifactual noise with high specificity and sensitivity. Noise cancelling recovered the composition of the mutational signatures in the tumor samples. Thus, we provide an informatics algorithm as a solution to the sequencing errors produced as a consequence of endonuclease-mediated fragmentation, highlighted for the first time in this study.

Retracted: Artifactual Palindrome and Mutations Resulting from the Stable Secondary Structure of Templates in Sanger Sequencing

“…In next-generation sequencing (NGS), investigations revealed that sequencing errors can be introduced in the process of DNA isolation, DNA fragmentation and library preparation, which interfered detection of rare variants. [20][21][22][23] In contrast, few studies revealed the sequence errors in sanger sequencing because the low-frequent substitutions were not considered at all, and the instrumentation and chemistry currently used for Sanger sequencing is highly reliable and reproducible. Thus, the unusual and intriguing sequencing artifacts in our study are deserved clear and detailed investigation to prevent the possible misinterpretation.…”

A Cell Culture Model of BK Polyomavirus Persistence, Genome Recombination, and Reactivation

“…We therefore sequenced BKPyV genomes over the course of our experiment and examined if recombination was occurring during persistent infection. We prepared DNA libraries directly from low-molecular-weight DNA with a transposon-based library preparation kit to minimize recombination artifacts that could be generated during the DNA library preparation ( 20 ). We also started with a maximum amount of the DNA template to minimize the number of PCR cycles required to barcode the library, such that we were able to prepare all libraries with only five PCR cycles.…”

Near-Complete Genome Sequence of a Human Norovirus GII.1[Pg] Strain Associated with Acute Gastroenteritis, Determined Using Long-Read Sequencing

“…In comparison with short-read sequencing, Nanopore long-read sequencing technologies make it possible to detect and identify viruses faster as well as accurately characterize the genomic complexity with complete coverage of the entire genome by a single read ( 6 ). In addition, as a fundamental step during library preparation in short-read sequencing, DNA fragmentation could introduce sequencing bias due to the randomness, incorporation errors, or artifactual indels caused by enzymatic effects ( 7 ), and these fragmentation biases can be lowered using Nanopore sequencing technology. Here, we report that using Nanopore sequencing technology and long reads, a nearly complete genome sequence was successfully obtained from a human norovirus GII.1[Pg] strain associated with a case of acute gastroenteritis in the United States in 2016.…”