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

Abstract

Multiple displacement amplification (MDA) has become one of the most commonly used method of whole genome amplification (WGA) due to the high processivity, strand displacement capacity and high fidelity of the phi29 DNA polymerase, MDA generate vast amount of DNA with higher molecules weight (up to 100kb) and greater genome coverage. Along with the development of the sequencing platform, it is possible to sequence the MDA-amplified DNA molecules with over 20kb by long-read sequencing. However, one of the challenges is the formation of chimeras, which exist in all MDA products, and seriously interfere with the downstream analysis of the long-read sequencing data of MDA-amplified DNA. In this study, we constructed 3rd-ChimeraMiner, a chimera detection pipeline for analyzing the long-read sequencing of MDA products, recognizing chimeras, and integrating chimeras into the downstream analysis. Five sequencing data of MDA with different magnification fold were analyzed in here, the proportions of chimeras are much higher than that of next-generation sequencing reads and increase with the increase of magnification folds, ranging from 42% to over 76%. After comparing, 99.92% of recognized chimeras have been demonstrated not to exist in original genomes. After detecting chimeras by 3rd-ChimeraMiner, the full-length mapping ratio increased, means more PacBio data could be used in downstream analysis, and mean 97.77% inversions were removed after transferred chimeras into normal reads. 3rd-ChimeraMiner revealed efficiency and accuracy in discovering chimeras from long-read sequencing data of MDA, and is promising to be widely used in single-cell sequencing.

Chimera: The spoiler in multiple displacement amplification

“…In this mini-review, we discuss the formation and systematic characteristics of MDA chimera [39] and present existing methodologies for detecting chimeras in MDA-treated short and long-read sequencing data from Sanger sequencing [38] , NGS [39] , [40] , [41] , [54] , and third-generation sequencing platforms [51] , [55] . We also provide information on the treatment of chimeras and their impact on the improvement of data utilization efficiency.…”

“… Yuya et al 2021 DNA Research [51] Phageomes PacBio Sequel 5551 0.33 1.83 LAST SACRA ∼ 630 Novel tool for pre-processing chimeras in PacBio reads and reducing chimeric rate, but small genome, no overlap sequence analysis, and no comprehensively exploration. Lu et al 2022 bioRxiv [55] Human PacBio Sequel 3821 3.55 13.57 minimap2 3rd-ChimeraMiner ∼ 168 Tool for analyzing chimeras in human long-read sequencing data and transferring the chimeras to normal reads, but DNA not from single cell just single cell level. …”

“…Recently, Lu et al detected and characterized chimeric reads in PacBio long-read sequencing reads by coupling MDA and PacBio single-molecule sequencing platform. They developed 3 rd -ChimeraMiner, a novel bioinformatics tool for recognizing and classifying MDA chimeras in PacBio long reads [55] . The tool first aligned PacBio reads to the hg19 reference genome using minimap2, and considered the mapped reads with an “SA” tag as segmented mapped reads (SMRs).…”

“…Sci. [41] 300 4.56% 152 Yuya et al 2021 DNA Research [51] 5551 72% 356 Lu et al 2022 bioRxiv [55] 3821 76.16% 469

Fig. 5 Distribution of chimeric rate versus the mean length of the sequence reads， including both inverted and direct chimeras.

…”

“…In PacBio metagenomic-sequencing data of MDA-amplified phageomes DNA [51] , an average of 72 % of PacBio reads were chimeras, which is unexpectedly high. In PacBio human-sequencing data [55] , the chimeric rates increased with the increasing amplification fold, ranging from 42 % (MDA products initiated from multi-cells, amplification fold of 10 2 ) to over 76 % (MDA products initiated from DNA amounts of the single-cell level, amplification fold of 10 6 ). The inverted chimera remained the dominant type of chimera (average 89%), consistent with the results of previous NGS analyses.…”