HaloPlex Targeted Resequencing for Mutation Detection in Clinical Formalin-Fixed, Paraffin-Embedded Tumor Samples
In recent years, the advent of massively parallel next-generation sequencing technologies has enabled substantial advances in the study of human diseases. Combined with targeted DNA enrichment methods, high sequence coverage can be obtained for different genes simultaneously at a reduced cost per sa...
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| Published in | The Journal of molecular diagnostics : JMD Vol. 17; no. 6; pp. 729 - 739 |
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| Main Authors | , , , , , , , , , |
| Format | Journal Article |
| Language | English |
| Published |
United States
Elsevier Inc
01.11.2015
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| Subjects | |
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| Abstract | In recent years, the advent of massively parallel next-generation sequencing technologies has enabled substantial advances in the study of human diseases. Combined with targeted DNA enrichment methods, high sequence coverage can be obtained for different genes simultaneously at a reduced cost per sample, creating unique opportunities for clinical cancer diagnostics. However, the formalin-fixed, paraffin-embedded (FFPE) process of tissue samples, routinely used in pathology departments, results in DNA fragmentation and nucleotide modifications that introduce a number of technical challenges for downstream biomolecular analyses. We evaluated the HaloPlex target enrichment system for somatic mutation detection in 80 tissue fractions derived from 20 clinical cancer cases with paired tumor and normal tissue available in both FFPE and fresh-frozen format. Several modifications to the standard method were introduced, including a reduced target fragment length and two strand capturing. We found that FFPE material can be used for HaloPlex-based target enrichment and next-generation sequencing, even when starting from small amounts of DNA. By specifically capturing both strands for each target fragment, we were able to reduce the number of false-positive errors caused by FFPE-induced artifacts and lower the detection limit for somatic mutations. We believe that the HaloPlex method presented here will be broadly applicable as a tool for somatic mutation detection in clinical cancer settings. |
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| AbstractList | In recent years, the advent of massively parallel next-generation sequencing technologies has enabled substantial advances in the study of human diseases. Combined with targeted DNA enrichment methods, high sequence coverage can be obtained for different genes simultaneously at a reduced cost per sample, creating unique opportunities for clinical cancer diagnostics. However, the formalin-fixed, paraffin-embedded (FFPE) process of tissue samples, routinely used in pathology departments, results in DNA fragmentation and nucleotide modifications that introduce a number of technical challenges for downstream biomolecular analyses. We evaluated the HaloPlex target enrichment system for somatic mutation detection in 80 tissue fractions derived from 20 clinical cancer cases with paired tumor and normal tissue available in both FFPE and fresh-frozen format. Several modifications to the standard method were introduced, including a reduced target fragment length and two strand capturing. We found that FFPE material can be used for HaloPlex-based target enrichment and next-generation sequencing, even when starting from small amounts of DNA. By specifically capturing both strands for each target fragment, we were able to reduce the number of false-positive errors caused by FFPE-induced artifacts and lower the detection limit for somatic mutations. We believe that the HaloPlex method presented here will be broadly applicable as a tool for somatic mutation detection in clinical cancer settings. In recent years, the advent of massively parallel next-generation sequencing technologies has enabled substantial advances in the study of human diseases. Combined with targeted DNA enrichment methods, high sequence coverage can be obtained for different genes simultaneously at a reduced cost per sample, creating unique opportunities for clinical cancer diagnostics. However, the formalin-fixed, paraffin-embedded (FFPE) process of tissue samples, routinely used in pathology departments, results in DNA fragmentation and nucleotide modifications that introduce a number of technical challenges for downstream biomotecular analyses. We evaluated the HaloPlex target enrichment system for somatic mutation detection in 80 tissue fractions derived from 20 clinical cancer cases with paired tumor and normal tissue available in both FFPE and fresh-frozen format. Several modifications to the standard method were introduced, including a reduced target fragment Length and two strand capturing. We found that FFPE material can be used for HaloPlex-based target enrichment and next-generation sequencing, even when starting from small amounts of DNA. By specifically capturing both strands for each target fragment, we were able to reduce the number of false-positive errors caused by FFPE-induced artifacts and Lower the detection limit for somatic mutations. We believe that the HaloPlex method presented here will be broadly applicable as a tool for somatic mutation detection in clinical cancer settings. |
| Author | Moens, Lotte N.J La Fleur, Linnéa Micke, Patrick Botling, Johan Falk-Sörqvist, Elin Ljungström, Viktor Sundström, Magnus Nilsson, Mats Mattsson, Johanna Mathot, Lucy |
| Author_xml | – sequence: 1 fullname: Moens, Lotte N.J – sequence: 2 fullname: Falk-Sörqvist, Elin – sequence: 3 fullname: Ljungström, Viktor – sequence: 4 fullname: Mattsson, Johanna – sequence: 5 fullname: Sundström, Magnus – sequence: 6 fullname: La Fleur, Linnéa – sequence: 7 fullname: Mathot, Lucy – sequence: 8 fullname: Micke, Patrick – sequence: 9 fullname: Nilsson, Mats – sequence: 10 fullname: Botling, Johan |
| BackLink | https://www.ncbi.nlm.nih.gov/pubmed/26354930$$D View this record in MEDLINE/PubMed https://urn.kb.se/resolve?urn=urn:nbn:se:su:diva-123776$$DView record from Swedish Publication Index https://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-269252$$DView record from Swedish Publication Index |
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| CitedBy_id | crossref_primary_10_3390_diagnostics12071539 crossref_primary_10_1245_s10434_019_07452_2 crossref_primary_10_1016_j_addr_2016_04_005 crossref_primary_10_1016_j_nbt_2015_10_003 crossref_primary_10_1021_acsomega_3c07472 crossref_primary_10_1016_j_lungcan_2019_01_003 crossref_primary_10_1080_03009734_2020_1826612 crossref_primary_10_1002_ijc_34880 crossref_primary_10_1016_j_fsigen_2021_102496 crossref_primary_10_53876_001c_72631 crossref_primary_10_1016_j_jtho_2020_05_019 crossref_primary_10_1136_esmoopen_2018_000442 crossref_primary_10_1186_s12575_018_0084_7 crossref_primary_10_1586_14737159_2016_1133298 crossref_primary_10_3390_jpm11070613 |
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| Title | HaloPlex Targeted Resequencing for Mutation Detection in Clinical Formalin-Fixed, Paraffin-Embedded Tumor Samples |
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