De novo sequence assembly requires bioinformatic checking of chimeric sequences

Abstract

De novo assembly of sequence reads from next generation sequencing platforms is a common strategy for detecting presence and sequencing of viruses in biospecimens. Amplification artifacts and presence of several related viruses in the same specimen can lead to assembly of erroneous, chimeric sequences. We now report that such chimeras can also occur between viral and non-viral biological sequences incorrectly joined together which may cause erroneous detection of viruses, highlighting the importance of performing a chimera checking step in bioinformatics pipelines. Using Illumina NextSeq and metagenomic sequencing, we analyzed 80 consecutive non-melanoma skin cancers (NMSCs) from 11 immunosuppressed patients together with 11 NMSCs from patients who had only developed 1 NMSC. We aligned high-quality reads against a Human Papillomavirus (HPV) database and found HPV sequences in 9/91 specimens. A previous bioinformatic analysis of the same crude sequencing data from some of these samples had found an additional 3 specimens to be HPV-positive after performing de novo assembly. The reason for the discrepancy was investigated and found to be mostly caused by chimeric sequences containing both viral and non-viral sequences. Non-viral sequences were present in these 3 samples. To avoid erroneous detection of HPV when performing sequencing, we thus developed a novel script to identify HPV chimeric sequences.

Ultrafast and Cost-Effective Pathogen Identification and Resistance Gene Detection in a Clinical Setting Using Nanopore Flongle Sequencing

“…Phenotypically, E. coli 125 was susceptible to penicillin ( Avershina et al, 2021b ), which suggests that the TEM-1B gene in this isolate was not expressed. Due to numerous rounds of PCR, Illumina sequencing data are prone to chimera generation ( Arroyo Mühr et al, 2020 ), and chimera removal is a common filtering step when using amplicon sequencing with Illumina ( Prodan et al, 2020 ). However, it seems doubtful that a TEM-1B-identical sequence would be chimera-generated.…”

Human Papillomavirus Detection by Whole-Genome Next-Generation Sequencing: Importance of Validation and Quality Assurance Procedures

“…Subsequent post assembly assessment is mandatory to reduce the risk of chimeric sequences and possible miscalling of HPV positivity in samples and/or erroneous calling of new HPV variants/genotypes. HPV-Chimera scripts exist to help researchers determine the accuracy of their HPV contigs [ 12 , 77 ].…”

“…Indeed, in recent years, various studies have utilized NGS approaches to generate detailed insights into potential disease-related mechanisms of HPV. NGS has shown that certain sublineages of HPV are associated with a higher risk of cancer [ 9 , 10 , 11 ], and its high sensitivity also allows the attributable fraction of cervical cancer associated with HPV to be determined with greater precision compared to traditional PCR techniques [ 12 , 13 ]. Further, NGS has identified certain single-nucleotide polymorphisms (SNPs) associated with a higher likelihood of viral persistence [ 14 ] and the key role of HPV E7 gene conservation in cervical cancer development [ 15 ].…”

CStone: A de novo transcriptome assembler for short-read data that identifies non-chimeric contigs based on underlying graph structure

“…The latter occurring during library preparation [6,7], or during the de novo assembly process [8,9], where there is a requirement to traverse paths across graphs constructed from read data that ranges in complexity depending on the nature of the gene families being represented [10][11][12]. Chimeras also occur at a genomic level during de novo assembly, such as when inferring haplotypes [13,14], but the causes, and consequences, at a genomic level are different [15][16][17]. In genomic assembly the aim is to reconstruct fewer large contigs that represent chromosomes [18,19].…”