Regulation of average length of complex PCR product

Shagin, Dmitry A.; Lukyanov, Konstantin A.; Vagner, Laura L.; Matz, Mikhail V.

doi:10.1093/nar/27.18.e23

Cited by 61 publications

(51 citation statements)

References 7 publications

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“…Primer mismatches have been shown, however, to influence relative abundance for other metabarcoding loci (Piñol et al., ). In addition, shorter molecules tend to amplify more readily than longer molecules during PCR (Shagin, Lukyanov, Vagner, & Matz, ), and while most sequences amplified by the trnL g/h primers in this study tended to be around the same length, other metabarcoding loci vary considerably in barcode length between amplified taxa. Another source of bias during PCR is homopolymer repeats (Kieleczawa, ).…”

Section: Discussionmentioning

confidence: 65%

Minimizing polymerase biases in metabarcoding

Nichols

Vollmers

Newsom

et al. 2018

Molecular Ecology Resources

182

175

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DNA metabarcoding is an increasingly popular method to characterize and quantify biodiversity in environmental samples. Metabarcoding approaches simultaneously amplify a short, variable genomic region, or "barcode," from a broad taxonomic group via the polymerase chain reaction (PCR), using universal primers that anneal to flanking conserved regions. Results of these experiments are reported as occurrence data, which provide a list of taxa amplified from the sample, or relative abundance data, which measure the relative contribution of each taxon to the overall composition of amplified product. The accuracy of both occurrence and relative abundance estimates can be affected by a variety of biological and technical biases. For example, taxa with larger biomass may be better represented in environmental samples than those with smaller biomass. Here, we explore how polymerase choice, a potential source of technical bias, might influence results in metabarcoding experiments. We compared potential biases of six commercially available polymerases using a combination of mixtures of amplifiable synthetic sequences and real sedimentary DNA extracts. We find that polymerase choice can affect both occurrence and relative abundance estimates and that the main source of this bias appears to be polymerase preference for sequences with specific GC contents. We further recommend an experimental approach for metabarcoding based on results of our synthetic experiments.

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Section: Discussionmentioning

confidence: 65%

Minimizing polymerase biases in metabarcoding

Nichols

Vollmers

Newsom

et al. 2018

Molecular Ecology Resources

182

175

View full text Add to dashboard Cite

show abstract

“…Genomic DNA was sheared by sonication, resulting in fragment sizes ranging from 1 kb to 100 bp. The ligation of PE 2 Walking Adapter to the fragments generates an intermediate library with two important characteristics: first, one adapter of the single strand template is identical to Illumina's PE PCR Primer 2 (PE 2); second, the adapter sequence is long enough to suppress unspecific amplification due to single-primer PCR effect [19] during the nested PCR. Targeted sequences were further enriched and full length Illumina PE sequencing adapters were introduced by PE PCR Primer 1 and 2 (PE 1 and PE 2).…”

Section: Resultsmentioning

confidence: 99%

Recurrent Targeted Genes of Hepatitis B Virus in the Liver Cancer Genomes Identified by a Next-Generation Sequencing–Based Approach

et al. 2012

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Integration of the viral DNA into host chromosomes was found in most of the hepatitis B virus (HBV)–related hepatocellular carcinomas (HCCs). Here we devised a massive anchored parallel sequencing (MAPS) method using next-generation sequencing to isolate and sequence HBV integrants. Applying MAPS to 40 pairs of HBV–related HCC tissues (cancer and adjacent tissues), we identified 296 HBV integration events corresponding to 286 unique integration sites (UISs) with precise HBV–Human DNA junctions. HBV integration favored chromosome 17 and preferentially integrated into human transcript units. HBV targeted genes were enriched in GO terms: cAMP metabolic processes, T cell differentiation and activation, TGF beta receptor pathway, ncRNA catabolic process, and dsRNA fragmentation and cellular response to dsRNA. The HBV targeted genes include 7 genes (PTPRJ, CNTN6, IL12B, MYOM1, FNDC3B, LRFN2, FN1) containing IPR003961 (Fibronectin, type III domain), 7 genes (NRG3, MASP2, NELL1, LRP1B, ADAM21, NRXN1, FN1) containing IPR013032 (EGF-like region, conserved site), and three genes (PDE7A, PDE4B, PDE11A) containing IPR002073 (3′, 5′-cyclic-nucleotide phosphodiesterase). Enriched pathways include hsa04512 (ECM-receptor interaction), hsa04510 (Focal adhesion), and hsa04012 (ErbB signaling pathway). Fewer integration events were found in cancers compared to cancer-adjacent tissues, suggesting a clonal expansion model in HCC development. Finally, we identified 8 genes that were recurrent target genes by HBV integration including fibronectin 1 (FN1) and telomerase reverse transcriptase (TERT1), two known recurrent target genes, and additional novel target genes such as SMAD family member 5 (SMAD5), phosphatase and actin regulator 4 (PHACTR4), and RNA binding protein fox-1 homolog (C. elegans) 1 (RBFOX1). Integrating analysis with recently published whole-genome sequencing analysis, we identified 14 additional recurrent HBV target genes, greatly expanding the HBV recurrent target list. This global survey of HBV integration events, together with recently published whole-genome sequencing analyses, furthered our understanding of the HBV–related HCC.

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“…10 7 cells or Ն1 g of poly(A) mRNA per routine hybridization] were needed (14,27). A variety of template amplification methods are currently under development to address this problem (e.g., linear amplification with T7 polymerase or template switching [41,42,65]). However, these methods may introduce a bias because of sequence-dependent processivity of the polymerase.…”

Section: Discussionmentioning

confidence: 99%

Charting Latency Transcripts in Kaposi's Sarcoma-Associated Herpesvirus by Whole-Genome Real-Time Quantitative PCR

Fakhari

Dittmer

2002

J Virol

193

219

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The division into a latent or lytic life cycle is fundamental to all herpesviridae. In the case of Kaposi's sarcoma-associated herpesvirus (KSHV) (human herpesvirus 8), latent genes have been implicated in cell autonomous transformation, while certain lytic genes procure a tumor friendly milieu through paracrine mechanism. To query KSHV transcription, we devised and validated a high-throughput, high-specificity, high-sensitivity, real-time quantitative reverse transcription-PCR array. This novel methodology is applicable to many human pathogens. Its first use demonstrated that the mRNA levels for KSHV LANA, v-cyclin, and v-FLIP do not increase at any time after viral reactivation. The mRNA for LANA-2/vIRF-3 is similarly resistant to viral reactivation. In contrast, every other latent or lytic message was induced. Hence, LANA, v-FLIP, v-cyclin, and LANA-2 constitute a group of uniquely regulated transcripts in the KSHV genome.

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Regulation of average length of complex PCR product

Cited by 61 publications

References 7 publications

Minimizing polymerase biases in metabarcoding

Minimizing polymerase biases in metabarcoding

Recurrent Targeted Genes of Hepatitis B Virus in the Liver Cancer Genomes Identified by a Next-Generation Sequencing–Based Approach

Charting Latency Transcripts in Kaposi's Sarcoma-Associated Herpesvirus by Whole-Genome Real-Time Quantitative PCR

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