ChimeraMiner: An Improved Chimeric Read Detection Pipeline and Its Application in Single Cell Sequencing

Abstract

As the most widely-used single cell whole genome amplification (WGA) approach, multiple displacement amplification (MDA) has a superior performance, due to the high-fidelity and processivity of phi29 DNA polymerase. However, chimeric reads, generated in MDA, cause severe disruption in many single-cell studies. Herein, we constructed ChimeraMiner, an improved chimeric read detection pipeline for analyzing the sequencing data of MDA and classified the chimeric sequences. Two datasets (MDA1 and MDA2) were used for evaluating and comparing the efficiency of ChimeraMiner and previous pipeline. Under the same hardware condition, ChimeraMiner spent only 43.4% (43.8% for MDA1 and 43.0% for MDA2) processing time. Respectively, 24.4 million (6.31%) read pairs out of 773 million reads, and 17.5 million (6.62%) read pairs out of 528 million reads were accurately classified as chimeras by ChimeraMiner. In addition to finding 83.60% (17,639,371) chimeras, which were detected by previous pipelines, ChimeraMiner screened 6,736,168 novel chimeras, most of which were missed by the previous pipeline. Applying in single-cell datasets, all three types of chimera were discovered in each dataset, which introduced plenty of false positives in structural variation (SV) detection. The identification and filtration of chimeras by ChimeraMiner removed most of the false positive SVs (83.8%). ChimeraMiner revealed improved efficiency in discovering chimeric reads, and is promising to be widely used in single-cell sequencing.

3rd-ChimeraMiner: A pipeline for integrated analysis of whole genome amplification generated chimeric sequences using long-read sequencing

“…And for different DNA concertation, the proportion of chimera is not consistent. For PacBio subread, the average chimeric ratio to be 76.16% for AF106 and 42.41% for AF102, indicating a considerably high chimeric ratio in PacBio subreads as observed previously 33–36, 53, 54 . With the increasing of the amplification fold, the chimeric rate and chimeric density increasing simultaneously (Figure 3).…”

“…As for the chimeric strand specificity, this phenomenon not only happened between two reverse strands, but also was observed on the same strand. Following the nomenclature of the previous research, fundamental chimerism could be divided into two types: inverted chimeras and direct chimeras 33, 36, 54 . Two subsections of inverted chimeras are aligned to two reverse strands, while two subsections of direct chimera could be aligned concordantly to one strand.…”

Facilitating the adoption of high‐throughput sequencing technologies as a plant pest diagnostic test in laboratories: A step‐by‐step description

“…Similarly, whole genome amplification techniques such as multiple displacement amplification commonly used in low‐input library preparation protocols for shotgun sequencing can produce chimeric sequences (Lasken & Stockwell, 2007; Quince et al, 2011). It is important to monitor and remove these sequences using appropriate tools before target identification (Anslan et al, 2018; Lu et al, 2019; Quince et al, 2011). Denoising/clustering (specific to metabarcoding) .…”

Dual UMIs and Dual Barcodes With Minimal PCR Amplification Removes Artifacts and Acquires Accurate Antibody Repertoire

“…Previous studies using sequential clone hybridization and Sanger sequencing revealed that over 30% of PCR products were chimeras introduced by template switching and PCRmediated recombination (17)(18)(19)(20)(21)(22)(23)(24). But these methods are not applicable for identifying and removing chimeras from Rep-seq data due to the high similarity of V, D, and J genes, and the extraordinary diversity of antibodies.…”

“…One of the major challenges of bulk Rep-seq is reducing artifactual sequences introduced by PCR amplification and HTS. Upon amplification of a mixture of similar sequences, a considerable number of chimeras, accounting for over 30% of all sequences, were introduced due to template switching and PCR-mediated recombination (17)(18)(19)(20)(21)(22)(23)(24), which substantially impacts our understanding of the antibody repertoire, including analyses of V gene assignment and SHM frequency (1), evaluations of clonal expansion and diversity, discovery of antigen-specific mAbs, and elucidation of the antibody maturation pathway. Therefore, the quantitative assessment of chimeras in Rep-seq data deserves serious attention, and their elimination is of great importance for extracting the most pertinent biological information from an antibody repertoire.…”