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Pacific Biosciences long range pcr
Barcoding schemes. Direct versus two‐step sample barcoding. A : In the direct barcoding scheme the sample specific barcodes, indicated by the blue and red for patients 1 and 2, respectively, are attached to the gene‐specific sequences (black arrows) and are introduced in a single <t>PCR</t> reaction. B : For the two‐step procedure for each individual, the region of interest is first amplified with a pair of gene‐specific primers with M13 forward (green) and reverse (purple) sequence tails. A symmetrical sample barcode, indicated by blue and red for patients 1 and 2, respectively, is introduced in a second PCR using a set of M13 barcode primers. A 5' padding sequence (black) is present on the index primers for both the direct and two‐step barcoding schemes to give all fragment identical end sequences to avoid ligation biases during the <t>SMRT</t> bell library preparation.
Long Range Pcr, supplied by Pacific Biosciences, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Average 86 stars, based on 1 article reviews
Price from $9.99 to $1999.99
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86/100 stars

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1) Product Images from "Flexible and Scalable Full‐Length CYP2D6 Long Amplicon PacBio Sequencing"

Article Title: Flexible and Scalable Full‐Length CYP2D6 Long Amplicon PacBio Sequencing

Journal: Human Mutation

doi: 10.1002/humu.23166

Barcoding schemes. Direct versus two‐step sample barcoding. A : In the direct barcoding scheme the sample specific barcodes, indicated by the blue and red for patients 1 and 2, respectively, are attached to the gene‐specific sequences (black arrows) and are introduced in a single PCR reaction. B : For the two‐step procedure for each individual, the region of interest is first amplified with a pair of gene‐specific primers with M13 forward (green) and reverse (purple) sequence tails. A symmetrical sample barcode, indicated by blue and red for patients 1 and 2, respectively, is introduced in a second PCR using a set of M13 barcode primers. A 5' padding sequence (black) is present on the index primers for both the direct and two‐step barcoding schemes to give all fragment identical end sequences to avoid ligation biases during the SMRT bell library preparation.
Figure Legend Snippet: Barcoding schemes. Direct versus two‐step sample barcoding. A : In the direct barcoding scheme the sample specific barcodes, indicated by the blue and red for patients 1 and 2, respectively, are attached to the gene‐specific sequences (black arrows) and are introduced in a single PCR reaction. B : For the two‐step procedure for each individual, the region of interest is first amplified with a pair of gene‐specific primers with M13 forward (green) and reverse (purple) sequence tails. A symmetrical sample barcode, indicated by blue and red for patients 1 and 2, respectively, is introduced in a second PCR using a set of M13 barcode primers. A 5' padding sequence (black) is present on the index primers for both the direct and two‐step barcoding schemes to give all fragment identical end sequences to avoid ligation biases during the SMRT bell library preparation.

Techniques Used: Polymerase Chain Reaction, Amplification, Sequencing, Ligation

Related Articles

Polymerase Chain Reaction:

Article Title: Loss of maternal EED results in postnatal overgrowth
Article Snippet: All data was collected using Lunar Piximus 2.0 software. .. Long-range PCR and sequencing of patient and parental DNA samples Regions spanning the defined mutations in the EZH2 gene of patients were amplified using long-range PCR in patient and parental DNA samples and processed for single molecule real-time (SMRT) sequencing (Pacific Bioscience) by the Leiden Genome Technology Centre, Department of Human Genetics, Leiden University Medical Center, the Netherlands. .. All samples were obtained by Professor William Gibson under University of British Columbia and British Columbia Children’s Hospital Human Ethics approval numbers H08-00784, H09-01228 and H10-03215, University of British Columbia, Vancouver, Canada.

Article Title: A hot L1 retrotransposon evades somatic repression and initiates human colorectal cancer
Article Snippet: Our analysis was focused on Human-specific (Hs) full-length elements (FL-L1Hs), since all active L1s in the human genome are found within this group. .. Using MELT and associated MELT tools, we identified a total of 308 FL-L1Hs elements in the patient's genome and analyzed the mutation profiles of these elements as follows (Methods): The full sequences of 264 reference FL-L1Hs copies were available in the hg19 human genome reference sequence ( ; ), and the sequences of 31 of 44 additional nonreference FL-L1Hs elements were determined using a combination of long-range PCR and Sanger or PacBio sequencing (Methods, ). .. By examining the internal mutation patterns of 295 of 308 (95.8%) of the FL-L1Hs elements compared to the L1.3 reference L1 element (GenBank ID ) , we learned that all but five of these elements had a unique singleton mutation that alone could be used to distinguish each individual element from all other FL-L1Hs elements in the patient's genome ( B,C; ).

Article Title: Pitfalls of haplotype phasing from amplicon-based long-read sequencing
Article Snippet: The long-read sequencers from Pacific Bioscience (PacBio) and Oxford Nanopore Technologies (ONT) offer the opportunity to phase mutations multiple kilobases apart directly from sequencing reads. .. In this study, we used long-range PCR with ONT and PacBio sequencing to phase two variants 9 kb apart in the RET gene. ..

Article Title: MON-319 A Young Woman with Familial Isolated Primary Hyperparathyroidism Due to Parathyroid Adenoma and Atrophic Gastritis
Article Snippet: .. Genetic test was negative for CASR, CDC73, CDKN1B, MEN1, RET amplified by long-range PCR and sequenced by Pacific Biosciences SMRT. ..

Article Title: Reference Grade Characterization of Polymorphisms in Full-Length HLA Class I and II Genes With Short-Read Sequencing on the ION PGM System and Long-Reads Generated by Single Molecule, Real-Time Sequencing on the PacBio Platform
Article Snippet: .. Less than one hundred minimum subreads were obtained in pool-3 that included long-range PCR amplicons of 6.1–11.2 kb, whereas over one hundred minimum subreads were obtained in pool-1 and pool-2 that included comparatively shorter PCR amplicons of 4.6–6.7 kb. ..

Article Title: CRISPR/Cas precision: do we need to worry about off-targeting in plants?
Article Snippet: .. Such chromosomal rearrangements are not always easy to detect unless long-range PCR or long-read next generation sequencing (NGS), such as PacBio, are used. .. Therefore, quite possibly, in many plant studies where targeted mutagenesis was performed using CRISPR/Cas, such unintended genomic changes might have remained undetected since the above-mentioned techniques were rarely used for genotyping CRISPR/Cas-induced mutations in plants.

Article Title: A new method for sequencing the hypervariable Plasmodium falciparum gene var2csa from clinical samples
Article Snippet: Targeted PacBio sequencing has been used to sequence complex loci in humans [ ], as well as in P. falciparum [ ]. .. This approach was modified here to develop a new sequencing assay for var2csa , which combines long-range PCR with PacBio sequencing and assembly. .. This amplicon sequencing of the var2csa N-terminal region captures approximately half of the full-length var2csa , including ID1-ID2a, the primary focus of the current VAR2CSA-based phase I vaccine trial.

Sequencing:

Article Title: Loss of maternal EED results in postnatal overgrowth
Article Snippet: All data was collected using Lunar Piximus 2.0 software. .. Long-range PCR and sequencing of patient and parental DNA samples Regions spanning the defined mutations in the EZH2 gene of patients were amplified using long-range PCR in patient and parental DNA samples and processed for single molecule real-time (SMRT) sequencing (Pacific Bioscience) by the Leiden Genome Technology Centre, Department of Human Genetics, Leiden University Medical Center, the Netherlands. .. All samples were obtained by Professor William Gibson under University of British Columbia and British Columbia Children’s Hospital Human Ethics approval numbers H08-00784, H09-01228 and H10-03215, University of British Columbia, Vancouver, Canada.

Article Title: A hot L1 retrotransposon evades somatic repression and initiates human colorectal cancer
Article Snippet: Our analysis was focused on Human-specific (Hs) full-length elements (FL-L1Hs), since all active L1s in the human genome are found within this group. .. Using MELT and associated MELT tools, we identified a total of 308 FL-L1Hs elements in the patient's genome and analyzed the mutation profiles of these elements as follows (Methods): The full sequences of 264 reference FL-L1Hs copies were available in the hg19 human genome reference sequence ( ; ), and the sequences of 31 of 44 additional nonreference FL-L1Hs elements were determined using a combination of long-range PCR and Sanger or PacBio sequencing (Methods, ). .. By examining the internal mutation patterns of 295 of 308 (95.8%) of the FL-L1Hs elements compared to the L1.3 reference L1 element (GenBank ID ) , we learned that all but five of these elements had a unique singleton mutation that alone could be used to distinguish each individual element from all other FL-L1Hs elements in the patient's genome ( B,C; ).

Article Title: Pitfalls of haplotype phasing from amplicon-based long-read sequencing
Article Snippet: The long-read sequencers from Pacific Bioscience (PacBio) and Oxford Nanopore Technologies (ONT) offer the opportunity to phase mutations multiple kilobases apart directly from sequencing reads. .. In this study, we used long-range PCR with ONT and PacBio sequencing to phase two variants 9 kb apart in the RET gene. ..

Article Title: A new method for sequencing the hypervariable Plasmodium falciparum gene var2csa from clinical samples
Article Snippet: Targeted PacBio sequencing has been used to sequence complex loci in humans [ ], as well as in P. falciparum [ ]. .. This approach was modified here to develop a new sequencing assay for var2csa , which combines long-range PCR with PacBio sequencing and assembly. .. This amplicon sequencing of the var2csa N-terminal region captures approximately half of the full-length var2csa , including ID1-ID2a, the primary focus of the current VAR2CSA-based phase I vaccine trial.

Amplification:

Article Title: Loss of maternal EED results in postnatal overgrowth
Article Snippet: All data was collected using Lunar Piximus 2.0 software. .. Long-range PCR and sequencing of patient and parental DNA samples Regions spanning the defined mutations in the EZH2 gene of patients were amplified using long-range PCR in patient and parental DNA samples and processed for single molecule real-time (SMRT) sequencing (Pacific Bioscience) by the Leiden Genome Technology Centre, Department of Human Genetics, Leiden University Medical Center, the Netherlands. .. All samples were obtained by Professor William Gibson under University of British Columbia and British Columbia Children’s Hospital Human Ethics approval numbers H08-00784, H09-01228 and H10-03215, University of British Columbia, Vancouver, Canada.

Article Title: MON-319 A Young Woman with Familial Isolated Primary Hyperparathyroidism Due to Parathyroid Adenoma and Atrophic Gastritis
Article Snippet: .. Genetic test was negative for CASR, CDC73, CDKN1B, MEN1, RET amplified by long-range PCR and sequenced by Pacific Biosciences SMRT. ..

Mutagenesis:

Article Title: A hot L1 retrotransposon evades somatic repression and initiates human colorectal cancer
Article Snippet: Our analysis was focused on Human-specific (Hs) full-length elements (FL-L1Hs), since all active L1s in the human genome are found within this group. .. Using MELT and associated MELT tools, we identified a total of 308 FL-L1Hs elements in the patient's genome and analyzed the mutation profiles of these elements as follows (Methods): The full sequences of 264 reference FL-L1Hs copies were available in the hg19 human genome reference sequence ( ; ), and the sequences of 31 of 44 additional nonreference FL-L1Hs elements were determined using a combination of long-range PCR and Sanger or PacBio sequencing (Methods, ). .. By examining the internal mutation patterns of 295 of 308 (95.8%) of the FL-L1Hs elements compared to the L1.3 reference L1 element (GenBank ID ) , we learned that all but five of these elements had a unique singleton mutation that alone could be used to distinguish each individual element from all other FL-L1Hs elements in the patient's genome ( B,C; ).

Next-Generation Sequencing:

Article Title: CRISPR/Cas precision: do we need to worry about off-targeting in plants?
Article Snippet: .. Such chromosomal rearrangements are not always easy to detect unless long-range PCR or long-read next generation sequencing (NGS), such as PacBio, are used. .. Therefore, quite possibly, in many plant studies where targeted mutagenesis was performed using CRISPR/Cas, such unintended genomic changes might have remained undetected since the above-mentioned techniques were rarely used for genotyping CRISPR/Cas-induced mutations in plants.

Modification:

Article Title: A new method for sequencing the hypervariable Plasmodium falciparum gene var2csa from clinical samples
Article Snippet: Targeted PacBio sequencing has been used to sequence complex loci in humans [ ], as well as in P. falciparum [ ]. .. This approach was modified here to develop a new sequencing assay for var2csa , which combines long-range PCR with PacBio sequencing and assembly. .. This amplicon sequencing of the var2csa N-terminal region captures approximately half of the full-length var2csa , including ID1-ID2a, the primary focus of the current VAR2CSA-based phase I vaccine trial.

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    Pacific Biosciences long range pcr
    Barcoding schemes. Direct versus two‐step sample barcoding. A : In the direct barcoding scheme the sample specific barcodes, indicated by the blue and red for patients 1 and 2, respectively, are attached to the gene‐specific sequences (black arrows) and are introduced in a single <t>PCR</t> reaction. B : For the two‐step procedure for each individual, the region of interest is first amplified with a pair of gene‐specific primers with M13 forward (green) and reverse (purple) sequence tails. A symmetrical sample barcode, indicated by blue and red for patients 1 and 2, respectively, is introduced in a second PCR using a set of M13 barcode primers. A 5' padding sequence (black) is present on the index primers for both the direct and two‐step barcoding schemes to give all fragment identical end sequences to avoid ligation biases during the <t>SMRT</t> bell library preparation.
    Long Range Pcr, supplied by Pacific Biosciences, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/long range pcr/product/Pacific Biosciences
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    long range pcr - by Bioz Stars, 2021-03
    86/100 stars
      Buy from Supplier

    86
    Pacific Biosciences long range pcr amplification
    Deletion breakpoint identification. The chromosome 7p14.3 genomic region is illustrated with tracks for the proband chromosomal microarray (CMA) results, genomic <t>PCR</t> mapping amplicon locations (1–9; green: amplified; red: did not amplify), and copy number variants detected among healthy individuals in the Database of Genomic Variants (DGV; blue: duplication; red: deletion) ( a ). Unambiguous breakpoint mapping was performed by long-read single molecule real-time <t>(SMRT)</t> sequencing (PacBio) of long-range PCR products that amplified across the deleted interval in the proband ( b ). These SMRT sequencing data were also aligned to a modified human genome reference that excluded the identified 72.8 kb deletion (chr7:33130616–33203409) ( c ), confirming that there were no other sequence alterations at the breakpoint locations. The precise deletion breakpoints were subsequently confirmed in the proband and both carrier parents by Sanger sequencing of the long-range PCR amplicons ( d )
    Long Range Pcr Amplification, supplied by Pacific Biosciences, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/long range pcr amplification/product/Pacific Biosciences
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    long range pcr amplification - by Bioz Stars, 2021-03
    86/100 stars
      Buy from Supplier

    86
    Pacific Biosciences sequencing long range pcr amplification
    Overview of treatments and <t>PacBio</t> results for patient 3. A ) The BCR-ABL1 IS% values measured by routine quantitative <t>RT-PCR</t> are shown in open circles. The sensitivity of this assay was measured for the BGUS reference gene and depicted by gray squares. As indicated by the red cross, the T315I mutation was detected after eleven months of imatinib treatment. The mutation was detected at this time point using our allele specific quantitative PCR used in routine analysis. The samples that were analyzed by PacBio sequencing are indicated by black arrows and their mutation composition showed in the circle plot diagrams. Vertical lines indicate the treatment periods. HU (Hydroxyurea). B) This panel shows the mutational composition in the BCR-ABL1 transcript for the 49 m and 55 m follow-up samples. Horizontal lines gives a schematic representation of high-quality PacBio reads that were used for examining the mutational composition. At 49 m, 91.8% of the reads carried T315I mutation. 4.2% of the reads showed the presence of F359C and 3.9% of the reads contained none of the mutations. At 55 m, two new clones appeared, one containing D276G and T315I (2.0% of the reads) and one containing T315I and H396R (1.1% of the reads).
    Sequencing Long Range Pcr Amplification, supplied by Pacific Biosciences, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/sequencing long range pcr amplification/product/Pacific Biosciences
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    sequencing long range pcr amplification - by Bioz Stars, 2021-03
    86/100 stars
      Buy from Supplier

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    Barcoding schemes. Direct versus two‐step sample barcoding. A : In the direct barcoding scheme the sample specific barcodes, indicated by the blue and red for patients 1 and 2, respectively, are attached to the gene‐specific sequences (black arrows) and are introduced in a single PCR reaction. B : For the two‐step procedure for each individual, the region of interest is first amplified with a pair of gene‐specific primers with M13 forward (green) and reverse (purple) sequence tails. A symmetrical sample barcode, indicated by blue and red for patients 1 and 2, respectively, is introduced in a second PCR using a set of M13 barcode primers. A 5' padding sequence (black) is present on the index primers for both the direct and two‐step barcoding schemes to give all fragment identical end sequences to avoid ligation biases during the SMRT bell library preparation.

    Journal: Human Mutation

    Article Title: Flexible and Scalable Full‐Length CYP2D6 Long Amplicon PacBio Sequencing

    doi: 10.1002/humu.23166

    Figure Lengend Snippet: Barcoding schemes. Direct versus two‐step sample barcoding. A : In the direct barcoding scheme the sample specific barcodes, indicated by the blue and red for patients 1 and 2, respectively, are attached to the gene‐specific sequences (black arrows) and are introduced in a single PCR reaction. B : For the two‐step procedure for each individual, the region of interest is first amplified with a pair of gene‐specific primers with M13 forward (green) and reverse (purple) sequence tails. A symmetrical sample barcode, indicated by blue and red for patients 1 and 2, respectively, is introduced in a second PCR using a set of M13 barcode primers. A 5' padding sequence (black) is present on the index primers for both the direct and two‐step barcoding schemes to give all fragment identical end sequences to avoid ligation biases during the SMRT bell library preparation.

    Article Snippet: Long‐Range PCR, SMRT Library Prep, and PacBio Sequencing All work described in this paper is subject to the LUMC Good Research Practice & Integrity guidelines and Ethical requirements.

    Techniques: Polymerase Chain Reaction, Amplification, Sequencing, Ligation

    Deletion breakpoint identification. The chromosome 7p14.3 genomic region is illustrated with tracks for the proband chromosomal microarray (CMA) results, genomic PCR mapping amplicon locations (1–9; green: amplified; red: did not amplify), and copy number variants detected among healthy individuals in the Database of Genomic Variants (DGV; blue: duplication; red: deletion) ( a ). Unambiguous breakpoint mapping was performed by long-read single molecule real-time (SMRT) sequencing (PacBio) of long-range PCR products that amplified across the deleted interval in the proband ( b ). These SMRT sequencing data were also aligned to a modified human genome reference that excluded the identified 72.8 kb deletion (chr7:33130616–33203409) ( c ), confirming that there were no other sequence alterations at the breakpoint locations. The precise deletion breakpoints were subsequently confirmed in the proband and both carrier parents by Sanger sequencing of the long-range PCR amplicons ( d )

    Journal: NPJ Genomic Medicine

    Article Title: Cytogenomic identification and long-read single molecule real-time (SMRT) sequencing of a Bardet–Biedl Syndrome 9 (BBS9) deletion

    doi: 10.1038/s41525-017-0042-3

    Figure Lengend Snippet: Deletion breakpoint identification. The chromosome 7p14.3 genomic region is illustrated with tracks for the proband chromosomal microarray (CMA) results, genomic PCR mapping amplicon locations (1–9; green: amplified; red: did not amplify), and copy number variants detected among healthy individuals in the Database of Genomic Variants (DGV; blue: duplication; red: deletion) ( a ). Unambiguous breakpoint mapping was performed by long-read single molecule real-time (SMRT) sequencing (PacBio) of long-range PCR products that amplified across the deleted interval in the proband ( b ). These SMRT sequencing data were also aligned to a modified human genome reference that excluded the identified 72.8 kb deletion (chr7:33130616–33203409) ( c ), confirming that there were no other sequence alterations at the breakpoint locations. The precise deletion breakpoints were subsequently confirmed in the proband and both carrier parents by Sanger sequencing of the long-range PCR amplicons ( d )

    Article Snippet: Long-read SMRT sequencing : Long-range PCR amplification across the identified breakpoint regions was accomplished using primers targeted to unique DNA sequences flanking the approximated deletion coordinates, and these amplicons were subjected to SMRTbell library construction and long-read SMRT sequencing (Pacific Biosciences, Menlo Park, CA).

    Techniques: Microarray, Polymerase Chain Reaction, Amplification, Sequencing, Modification

    Deletion breakpoint identification. The chromosome 7p14.3 genomic region is illustrated with tracks for the proband chromosomal microarray (CMA) results, genomic PCR mapping amplicon locations (1–9; green: amplified; red: did not amplify), and copy number variants detected among healthy individuals in the Database of Genomic Variants (DGV; blue: duplication; red: deletion) ( a ). Unambiguous breakpoint mapping was performed by long-read single molecule real-time (SMRT) sequencing (PacBio) of long-range PCR products that amplified across the deleted interval in the proband ( b ). These SMRT sequencing data were also aligned to a modified human genome reference that excluded the identified 72.8 kb deletion (chr7:33130616–33203409) ( c ), confirming that there were no other sequence alterations at the breakpoint locations. The precise deletion breakpoints were subsequently confirmed in the proband and both carrier parents by Sanger sequencing of the long-range PCR amplicons ( d )

    Journal: NPJ Genomic Medicine

    Article Title: Cytogenomic identification and long-read single molecule real-time (SMRT) sequencing of a Bardet–Biedl Syndrome 9 (BBS9) deletion

    doi: 10.1038/s41525-017-0042-3

    Figure Lengend Snippet: Deletion breakpoint identification. The chromosome 7p14.3 genomic region is illustrated with tracks for the proband chromosomal microarray (CMA) results, genomic PCR mapping amplicon locations (1–9; green: amplified; red: did not amplify), and copy number variants detected among healthy individuals in the Database of Genomic Variants (DGV; blue: duplication; red: deletion) ( a ). Unambiguous breakpoint mapping was performed by long-read single molecule real-time (SMRT) sequencing (PacBio) of long-range PCR products that amplified across the deleted interval in the proband ( b ). These SMRT sequencing data were also aligned to a modified human genome reference that excluded the identified 72.8 kb deletion (chr7:33130616–33203409) ( c ), confirming that there were no other sequence alterations at the breakpoint locations. The precise deletion breakpoints were subsequently confirmed in the proband and both carrier parents by Sanger sequencing of the long-range PCR amplicons ( d )

    Article Snippet: Long-read SMRT sequencing : Long-range PCR amplification across the identified breakpoint regions was accomplished using primers targeted to unique DNA sequences flanking the approximated deletion coordinates, and these amplicons were subjected to SMRTbell library construction and long-read SMRT sequencing (Pacific Biosciences, Menlo Park, CA).

    Techniques: Microarray, Polymerase Chain Reaction, Amplification, Sequencing, Modification

    Overview of treatments and PacBio results for patient 3. A ) The BCR-ABL1 IS% values measured by routine quantitative RT-PCR are shown in open circles. The sensitivity of this assay was measured for the BGUS reference gene and depicted by gray squares. As indicated by the red cross, the T315I mutation was detected after eleven months of imatinib treatment. The mutation was detected at this time point using our allele specific quantitative PCR used in routine analysis. The samples that were analyzed by PacBio sequencing are indicated by black arrows and their mutation composition showed in the circle plot diagrams. Vertical lines indicate the treatment periods. HU (Hydroxyurea). B) This panel shows the mutational composition in the BCR-ABL1 transcript for the 49 m and 55 m follow-up samples. Horizontal lines gives a schematic representation of high-quality PacBio reads that were used for examining the mutational composition. At 49 m, 91.8% of the reads carried T315I mutation. 4.2% of the reads showed the presence of F359C and 3.9% of the reads contained none of the mutations. At 55 m, two new clones appeared, one containing D276G and T315I (2.0% of the reads) and one containing T315I and H396R (1.1% of the reads).

    Journal: BMC Cancer

    Article Title: Clonal distribution of BCR-ABL1 mutations and splice isoforms by single-molecule long-read RNA sequencing

    doi: 10.1186/s12885-015-1046-y

    Figure Lengend Snippet: Overview of treatments and PacBio results for patient 3. A ) The BCR-ABL1 IS% values measured by routine quantitative RT-PCR are shown in open circles. The sensitivity of this assay was measured for the BGUS reference gene and depicted by gray squares. As indicated by the red cross, the T315I mutation was detected after eleven months of imatinib treatment. The mutation was detected at this time point using our allele specific quantitative PCR used in routine analysis. The samples that were analyzed by PacBio sequencing are indicated by black arrows and their mutation composition showed in the circle plot diagrams. Vertical lines indicate the treatment periods. HU (Hydroxyurea). B) This panel shows the mutational composition in the BCR-ABL1 transcript for the 49 m and 55 m follow-up samples. Horizontal lines gives a schematic representation of high-quality PacBio reads that were used for examining the mutational composition. At 49 m, 91.8% of the reads carried T315I mutation. 4.2% of the reads showed the presence of F359C and 3.9% of the reads contained none of the mutations. At 55 m, two new clones appeared, one containing D276G and T315I (2.0% of the reads) and one containing T315I and H396R (1.1% of the reads).

    Article Snippet: Library preparation and PacBio sequencing Long range PCR amplification of the BCR-ABL1 p210 transcript was performed using the Clontech Advantage PCR kit (Clontech, CA, USA).

    Techniques: Quantitative RT-PCR, Mutagenesis, Real-time Polymerase Chain Reaction, Sequencing, Clone Assay

    Overview of treatments and PacBio results for patients with single mutations. A) Results for patient 1. The BCR-ABL1 IS% values measured by routine quantitative RT-PCR are shown in open circles. The sensitivity of this assay was measured for the BGUS reference gene and depicted by gray squares. The samples that were analyzed by PacBio sequencing are indicated by black arrows and their mutation composition showed in the circle plot diagrams above each time point. Vertical lines indicate the treatment periods. HU (Hydroxyurea). B) Results for patient 2. The T315I mutation was detected after nilotinib treatment, as indicated by the red cross. The mutation was detected at this time point using our allele specific quantitative PCR used in routine analysis.

    Journal: BMC Cancer

    Article Title: Clonal distribution of BCR-ABL1 mutations and splice isoforms by single-molecule long-read RNA sequencing

    doi: 10.1186/s12885-015-1046-y

    Figure Lengend Snippet: Overview of treatments and PacBio results for patients with single mutations. A) Results for patient 1. The BCR-ABL1 IS% values measured by routine quantitative RT-PCR are shown in open circles. The sensitivity of this assay was measured for the BGUS reference gene and depicted by gray squares. The samples that were analyzed by PacBio sequencing are indicated by black arrows and their mutation composition showed in the circle plot diagrams above each time point. Vertical lines indicate the treatment periods. HU (Hydroxyurea). B) Results for patient 2. The T315I mutation was detected after nilotinib treatment, as indicated by the red cross. The mutation was detected at this time point using our allele specific quantitative PCR used in routine analysis.

    Article Snippet: Library preparation and PacBio sequencing Long range PCR amplification of the BCR-ABL1 p210 transcript was performed using the Clontech Advantage PCR kit (Clontech, CA, USA).

    Techniques: Quantitative RT-PCR, Sequencing, Mutagenesis, Real-time Polymerase Chain Reaction

    Overview of treatments and PacBio results for patients 4 and 5. A) Results for patient 4. The BCR-ABL1 IS% values measured by routine quantitative RT-PCR are shown in open circles. The sensitivity of this assay was measured for the BGUS reference gene and depicted by gray squares. As indicated by the red cross, the T315I mutation was detected after 58 months of dasatinib treatment. The mutation was detected at this time point using our allele specific quantitative PCR used in routine analysis. The samples that were analyzed by PacBio sequencing are indicated by black arrows and their mutation composition showed in the circle plot diagrams. Vertical lines indicate the treatment periods. HU (Hydroxyurea). B) Results for patient 5. Measurements shown were made as in A. AlloSCT: allogeneic stem cell transplantation. DLI: donor lymphocyte infusion.

    Journal: BMC Cancer

    Article Title: Clonal distribution of BCR-ABL1 mutations and splice isoforms by single-molecule long-read RNA sequencing

    doi: 10.1186/s12885-015-1046-y

    Figure Lengend Snippet: Overview of treatments and PacBio results for patients 4 and 5. A) Results for patient 4. The BCR-ABL1 IS% values measured by routine quantitative RT-PCR are shown in open circles. The sensitivity of this assay was measured for the BGUS reference gene and depicted by gray squares. As indicated by the red cross, the T315I mutation was detected after 58 months of dasatinib treatment. The mutation was detected at this time point using our allele specific quantitative PCR used in routine analysis. The samples that were analyzed by PacBio sequencing are indicated by black arrows and their mutation composition showed in the circle plot diagrams. Vertical lines indicate the treatment periods. HU (Hydroxyurea). B) Results for patient 5. Measurements shown were made as in A. AlloSCT: allogeneic stem cell transplantation. DLI: donor lymphocyte infusion.

    Article Snippet: Library preparation and PacBio sequencing Long range PCR amplification of the BCR-ABL1 p210 transcript was performed using the Clontech Advantage PCR kit (Clontech, CA, USA).

    Techniques: Quantitative RT-PCR, Mutagenesis, Real-time Polymerase Chain Reaction, Sequencing, Transplantation Assay

    Overview of treatments and PacBio results for patient 6. A) BCR-ABL1 IS% values measured by routine quantitative RT-PCR are shown in open circles. The sensitivity of this assay was measured for the BGUS reference gene and depicted by gray squares. The samples that were analyzed by PacBio sequencing are indicated by black arrows. The vertical line indicates the treatment period. B) This panel shows BCR-ABL1 isoforms in patient 6. At 7 months post diagnosis four different splice isoforms were identified. The most common isoform was the ‘wild type’ (WT) BCR-ABL1 transcript isoform, i.e. identical to the reference sequence used for mapping, present in 80% of the molecules. Two other isoforms contained insertions of entire exons, of lengths 35 bp and 154 bp, respectively and one contained a partial deletion of exon 7 of ABL1 . At 13 months post diagnosis the WT isoform was present in 54% of the molecules whereas isoforms containing the 35 bp insertion between exon 8 and 9 in ABL1 was present in the other two isoforms.

    Journal: BMC Cancer

    Article Title: Clonal distribution of BCR-ABL1 mutations and splice isoforms by single-molecule long-read RNA sequencing

    doi: 10.1186/s12885-015-1046-y

    Figure Lengend Snippet: Overview of treatments and PacBio results for patient 6. A) BCR-ABL1 IS% values measured by routine quantitative RT-PCR are shown in open circles. The sensitivity of this assay was measured for the BGUS reference gene and depicted by gray squares. The samples that were analyzed by PacBio sequencing are indicated by black arrows. The vertical line indicates the treatment period. B) This panel shows BCR-ABL1 isoforms in patient 6. At 7 months post diagnosis four different splice isoforms were identified. The most common isoform was the ‘wild type’ (WT) BCR-ABL1 transcript isoform, i.e. identical to the reference sequence used for mapping, present in 80% of the molecules. Two other isoforms contained insertions of entire exons, of lengths 35 bp and 154 bp, respectively and one contained a partial deletion of exon 7 of ABL1 . At 13 months post diagnosis the WT isoform was present in 54% of the molecules whereas isoforms containing the 35 bp insertion between exon 8 and 9 in ABL1 was present in the other two isoforms.

    Article Snippet: Library preparation and PacBio sequencing Long range PCR amplification of the BCR-ABL1 p210 transcript was performed using the Clontech Advantage PCR kit (Clontech, CA, USA).

    Techniques: Quantitative RT-PCR, Sequencing