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Translational Research | Citation
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Translational Research | Citations

Targeted Resequencing

The following are examples of peer-reviewed studies using either TaKaRa LA Taq HS DNA Polymerase or PrimeStar GXL DNA Polymerase for targeted resequencing:

Upstream NGS platform Target Disease state studied Polymerase Ref.
HiSeq® 2500 (Illumina®) MYH9 MYH9 disorders (bleeding disorder) TaKaRa LA Taq HS 1
Genome Analyzer® II (Illumina) RET protooncogene Cancer (and proof-of-concept technique study) TaKaRa LA Taq HS 2
Genome Analyzer II (Illumina) and Roche 454 GS FLX 9 USH loci Usher syndrome TaKaRa LA Taq HS 3
HiSeq 2000 (Illumina) Entire mtDNA genome Mitochondrial diseases TaKaRa LA Taq HS 4
MiSeq® (Illumina) TET2, IDH1, and IDH2 Myelodysplastic syndrome TaKaRa LA Taq HS 5
HiSeq 2000 (Illumina) 8 candidate genes identified by whole exome sequencing Juvenile myelomonocytic leukemia (JMML) PrimeSTAR GXL 6
HiSeq 2000 (Illumina Mutations identified in iPSC lines Point mutations associated with induced pluripotent stem cell (iPSC) generation PrimeSTAR GXL 7
MiSeq (Illumina) 6 different HLA genes Over 100 disease states are associated with the HLA region PrimeSTAR GXL 8
PacBio RS (Pacific Biosciences) Complex repetitive region of A. phagocytophilum virB6-4 Granulocytic anaplasmosis caused by a tick-borne pathogen, A. phagocytophilum PrimeSTAR GXL 9
454 GS-FLX (Roche) NLRP3 Neonatal onset multisystem inflammatory disease (NOMID) PrimeSTAR GXL 10
Genome Sequencer FLX (Roche) 16S rRNA Microbes in blood and organs of drowning victims PrimeSTAR GXL 11
GS Junior System (Roche), Ion Torrent PGM (Life Technologies) Multiple HLA loci (e.g., DRB1, DRB2, DRB3 Over 100 disease states are associated with HLA region PrimeSTAR GXL 12
(two refs)
  1. Kitamura, K., et al. (2013) Normal neutrophil myosin IIA localization in an immunofluorescence analysis can rule out MYH9 disorders. J. Thrombosis Haemostasis 11:2071–2073.

    Summary: Mutations in the MYH9 gene, which encodes non-muscle myosin heavy chain IIA (NMMHC-IIA), can result in mild to moderate bleeding tendencies and in some cases progressive nephritis, cataracts, and sensorineural hearing loss. MYH9 disorders are often diagnosed by the presence of NMMHC-IIA protein aggregates as assessed by peripheral blood smear staining using May-Giemsa stain or by more sensitive NMMHC-IIA immunofluorescence analysis techniques. To assess whether negative NMMHC-IIA immunofluorescence results correlate with absence of MYH9 gene mutations, the authors performed targeted deep resequencing on DNA from peripheral blood samples of 39 suspected congenital macrothrombocytopenia patients who had negative results for the immunofluorescence test. The entire coding sequence of MYH9 was amplified using TaKaRa LA Taq HS, and pooled samples were fragmented, prepared, and subsequently sequenced using a HiSeq 2500 sequencer (Illumina). No known pathognomonic MYH9 mutation nor any previously unreported sequence variant was detected.

  2. Margraf, R.L., et al. (2010) Multi-sample pooling and Illumina Genome Analyzer sequencing. Methods to determine gene sequence variation for database development. J. Biomol. Tech. 21(3):126–140.

    Summary: To develop methods for determining sequence variation within a genetic locus, researchers analyzed a pool of 10 samples with 13 known unique variants in the RET proto-oncogene on an Illumina Genome Analyzer (GA) system. TaKaRa LA Taq HS DNA Polymerase was used to amplify an 8868-bp amplicon spanning the RET gene from intron 9 to intron 16, and the amplicons were used for library prep and NGS analysis. The authors suggested that the “methods presented in this report could be used for any gene or small genome, where nonbar-coded, multiple sample pooling on the Illumina GA is used to determine population sequence variation.”

  3. Licastro, D., et al. (2012) Molecular diagnosis of Usher Syndrome: Application of two different Next Generation Sequencing-based procedures. PLoS ONE 7(8): e43799.

    Summary: Usher syndrome is a rare disorder that results from mutation of any one of 9 genes. The authors describe NGS approaches for genetic analysis of Usher syndrome patient genomic DNA samples in a comparison of whole exome sequencing versus targeted resequencing of long amplicons. TaKaRa LA Taq HS was used for long-range PCR upstream of NGS library preparation followed by targeted resequencing with either Illumina GAII or Roche 454 GS FLX platforms.

  4. Zhang, W., et al. (2012) Comprehensive one-step molecular analyses of mitochondrial genome by massively parallel sequencing. Clinical Chemistry 58(9):1322–1331.

    Summary: Mitochondrial disorders are clinically and genetically heterogeneous, with variable penetrance, expressivity, and age of onset. Deleterious mtDNA mutations are usually present in a heteroplasmic state, and the degree of heteroplasmy determines clinical severity. This heteroplasmy makes clinical diagnosis of mtDNA mutations technically challenging, prompting research into new techniques. The authors reported a one-step massively parallel sequencing technique to provide quantitative base calls, exact deletion junction sequences, and quantification of deletion heteroplasmy. TaKaRa LA Taq HS polymerase was used for long range PCR to amplify the entire mitochondrial genome from genomic DNA extracted from blood or skeletal muscle. Samples were then fragmented and libraries were prepared, enriched, and sequenced using an Illumina HiSeq 2000 system.

  5. Lin, T-L., et al. (2014) Clonal leukemic evolution in myelodysplastic syndromes with TET2 and IDH1/2 mutations. Haematologica 99(1):28–36.

    Summary: Myelodysplastic syndromes (MDS) are highly heterogeneous clonal hematologic malignancies associated with an increased risk of secondary acute myeloid leukemia (sAML). Numerous mutations have been associated with MDS, including mutations in TET2 (loss-of-function mutations in TET2 result in dysregulated DNA methylation) and in the isocitrate dehydrogenase-encoding genes, IDH1 and IDH2. To clarify the role of TET2, IDH1, and IDH2 mutations in progression to sAML, the authors examined TET2 and IDH mutations in a large cohort of de novo MDS cases including a large number (n=46) of paired MDS/sAML samples. TaKaRa LA Taq HS polymerase was used for PCR amplification of 46 genomic fragments harboring each target mutation of interest, followed by pooling and preparation for random deep sequencing using a MiSeq system (Illumina). The authors found that while TET2 and IDH mutations did not have any significant impact on overall survival, TET2 mutations were significantly associated with development of sAML.

  6. Sakaguchi, H., et al. (2013) Exome sequencing identifies secondary mutations of SETBP1 and JAK3 in juvenile myelomonocytic leukemia. Nature Genetics 45:937–941.

    Summary: Juvenile myelomonocytic leukemia (JMML) is a pediatric leukemia with poor prognosis and poorly understood molecular etiology. Somatic and/or germline mutations of RAS pathway members occur in >70% of JMML cases. However, more information is needed about whether these mutations are sufficient to initiate JMML, or whether secondary mutations influence disease development and progression. The authors performed whole-exome sequencing on paired tumor-normal DNA samples from 13 individuals with JMML. Somatic mutations identified included one nonsense and 10 missense mutations; of these, six involved known RAS pathway genes. Novel non-RAS pathway genes included SETBP1, JAK3, and SH3BP1. Researchers then performed targeted deep sequencing covering all exons of 8 target genes in 92 tumor samples using PrimeSTAR GXL polymerase for amplification. Amplicons were prepared and sequenced on a HiSeq 2000 system (Illumina). Mutations in SETBP1 and JAK3 were common secondary events that were associated with poor survival.

  7. Sugiura, M., et al. (2013) Induced pluripotent stem cell generation-associated point mutations arise during the initial stages of the conversion of these cells. Stem Cell Reports 2:52–63.

    Summary: While the presence of point mutations in induced pluripotent stem cells (iPSCs) has been widely reported, the origin of such mutations has been a matter of much debate. Could the mutations be age-accrued, preexisting single nucleotide variations (SNVs) present in parent somatic cells? Or are they newly introduced during reprogramming processes? To investigate these possibilities, researchers established new embryonic stem cell (ESC) lines and genome-integration-free iPSC lines and performed comparative whole-genome sequencing to identify candidate point mutations. Ultra-deep targeted resequencing was performed on 26 of the mutations identified in the iPS136 genome. These mutations were chosen at random to assess their presence in the parent somatic cells; only one of 26 did occur in parental cells, suggesting de novo mutations occurred during reprogramming. PrimeSTAR GXL polymerase was used for amplification, followed by ligation of mixed PCR products to Illumina paired-end sequencing adaptors and sequencing on the HiSeq 2000 system. Further study included variant allele frequency analysis performed with Titanium Taq DNA Polymerase and an Ion Torrent PGM sequencer.

  8. Hosomichi, K., et al. (2013) Phase-defined complete sequencing of the HLA genes by Next-Generation Sequencing. BMC Genomics 14:355.

    Summary: The highly polymorphic HLA region on chromosome 6 comprises 0.13% of the human genome but is associated with over 100 different diseases, including type I diabetes, rheumatoid arthritis, asthma, and psoriasis as well hypersensitivity to several different drugs. Conventional methods of HLA genotyping suffer from ambiguity or limited ability to detect novel alleles, whereas NGS approaches have been unable to attain full sequencing of the HLA region due to its extraordinarily complex and polymorphic nature. The authors developed a new technique for NGS-based HLA sequencing, relying on PrimeSTAR GXL polymerase for long-range PCR amplification of six HLA genes (HLA-A, -C, -B, -DRB1, -DQB1, and -DPB1). Product sizes ranged in length from 3,398 to 13,605 bp. The PCR products were used for transposase-based library construction using the Nextera DNA Sample Prep Kit, followed by sequencing on a MiSeq system (Illumina). Researchers reported that this approach generated phase-defined sequences of entire HLA genes, allowing both new allele detection and high resolution HLA typing.

  9. Al-Khedery, B., et al. (2012) Structure of the type IV secretion system in different strains of Anaplasma phagocytophilum. BMC Genomics 13:678.

    Summary: The potentially fatal human disease granulocytic anaplasmosis manifests as flu-like illness accompanied by leukopenia, thrombocytopenia and anemia, and is thought to be caused by the tick-borne pathogen Anaplasma phagocytophilum. A. phagocytophilum possesses a Type 4 Secretion System (T4SS) that secretes virulence (vir) factors affecting host immunity and pathogen responses, but the genetic structure of certain vir loci has been difficult to study due to their size and complexity. Using an NGS approach, the authors determined the structure of the T4SS in 7 strains from the U.S. and Europe and revised the sequence of the repetitive virB6 locus of the human HZ strain. PrimeSTAR GXL polymerase was used to amplify regions of the virB6-4 locus that include hypervariable and repetitive sequences. Products were cloned and subjected to sequencing using a long-read PacBio RS system (Pacific Biosciences).

  10. Izawa, K., et al. (2012) Detection of base substitution-type somatic mosaicism of the NLRP3 gene with >99.9% statistical confidence by massively parallel sequencing. DNA Res. 19(2):143–152.

    Summary: Neonatal onset multisystem inflammatory disease (NOMID) is a rare genetic syndrome that causes uncontrolled inflammation in multiple parts of the body, with symptoms beginning in infancy. NOMID is initiated by either germline or somatic mutation in the NLRP3 gene. Because rapid diagnosis of somatic mosaicism is critical for appropriate treatment, Izawa and colleagues aimed to develop a strategy to detect low-level alleles of NLRP3 using massively parallel DNA sequencing (MPS). PrimeSTAR GXL DNA Polymerase was used to prepare NLRP3 gene amplicons for MPS on a Roche 454 GS-FLX sequencer. The authors note that although the de novo somatic mutation rate is generally lower than the error rate of high-fidelity DNA polymerases used to generate the amplicons, using PrimeSTAR GXL polymerase, PCR-generated mosaicism higher than 1% was not detected in the sequencing error maps. Thus, with this sequencing strategy, base substitutions in the NLRP3 gene with allele frequencies as low as 1% could be detected with ≥99.9% confidence.

  11. Kakizaki, E., et al. (2012) Detection of diverse aquatic microbes in blood and organs of drowning victims: First metagenomic approach using high-throughput 454-pyrosequencing. Forensic Sci. 220:135–146.

    Summary: A key problem in forensic science is determining drowning as a cause of death. One approach is to analyze microbes in lungs, bloodstream, and organs and make comparisons to aquatic microbes found in a body of water. Some aquatic microbes (including bacteria, cyanobacteria, and eukaryotes such as diatoms and algae) can be absorbed from lungs into the bloodstream, suggesting aspiration prior to death. Classical analyses rely on culturing techniques, but better, faster, and more sensitive methods are needed. The authors developed an NGS technique for microbial profiling using primers that amplify conserved sequence flanking variable regions V7 and V8 of 16S microbial rRNA genes. The primers included adaptors and multiplex identifiers for pyrosequencing on a GS FLX Titanium system (Roche). DNA samples were prepared from blood, organs, and water of two cadavers and PrimeSTAR GXL polymerase was used for PCR, followed by pyrosequencing. Whereas conventional culture-based methods had supported drowning as a cause of death for one case but was inconclusive for the other, pyrosequencing metagenomic analysis enabled detection of aquatic microbes in blood of both bodies. The authors reported that the pyrosequencing approach yielded far more information about microbial populations while requiring less time and far less tissue than conventional techniques.

  12. Shiina, T., et al. (2012) Super high resolution for single molecule-sequence-based typing of classical HLA loci at the 8-digit level using next generation sequencers. Tissue Antigens 80:305–316.
    Ozaki, Y., et al. (2014) HLA-DRB1, -DRB3, -DRB4 and -DRB5 genotyping at a super-high resolution level by long range PCR and high-throughput sequencing. Tissue Antigens 83:10–16.

    Summary: To facilitate polymorphism discovery within the entire HLA region, the authors developed a system for single molecule sequence-based typing (SS-SBT) by combining long-range PCR with NGS. In an initial proof-of-concept publication (Shiina et al., 2012), researchers used PrimeSTAR GXL polymerase for long-range PCR amplification of the entire gene sequence—from enhancer-promoter region to the 3' UTR region—of eight HLA loci. PCR amplicons for each locus were sequenced individually using a Roche GS Junior system, allowing HLA typing at the 8-digit level (field 4 level) in both phases. Mixed PCR products were also used to construct barcoded libraries for sequencing on an Ion Torrent PGM system. In a later study, Ozaki and colleagues further refined the technique for characterizing DRB1-DLB3/ 4/ 5 haplotypes. The authors stated, “An important component for genotyping HLA by NGS in our study was the use of a single, high fidelity DNA polymerase product (TaKaRa Bio, Japan) in the PCR method that accurately and effectively amplifies PCR products of more than 4 kb from the genomic DNA samples.”

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