cfdfde2c64658b7d9626072dbb933870 biomedicines-11-03222.pdf 6aa274e8fedc457a71fb3095d4d71883a0a69fc3 biomedicines-11-03222.pdf eb56625903e5e681c3955e473ca3ba2727fdd92f69b6626b95273a718dc1b041 biomedicines-11-03222.pdf Title: Two-Time Multiplexed Targeted Next-Generation Sequencing Might Help the Implementation of Germline Screening Tools for Myelodysplastic Syndromes/Hematologic Neoplasms Subject: Next-generation sequencing (NGS) tools have importantly helped the classification of myelodysplastic syndromes (MDS), guiding the management of patients. However, new concerns are under debate regarding their implementation in routine clinical practice for the identification of germline predisposition. Cost-effective targeted NGS tools would improve the current standardized studies and genetic counseling. Here, we present our experience in a preliminary study detecting variants using a two-time multiplexed library strategy. Samples from different MDS patients were first mixed before library preparation and later multiplexed for a sequencing run. Two different mixes including a pool of three (3) and four (4) samples were evaluated. The filtered variants found in the individually sequenced samples were compared with the variants found in the two-time multiplexed studies to determine the detection efficiency scores. The same candidate variants were found in the two-time multiplexed studies in comparison with the individual tNGS. The variant allele frequency (VAF) values of the candidate variants were also compared. No significant differences were found between the expected and observed VAF percentages in both the 3 (p-value 0.74) and 4 (p-value 0.34) multiplexed studies. Our preliminary results suggest that the two-time multiplexing strategy might have the potential to help reduce the cost of evaluating germline predisposition. Keywords: myelodysplastic syndromes (MDS); genetic predisposition; targeted next-generation sequencing (tNGS); germline variants; genomic screening; multiplexed samples Author: Oriol Calvete, Julia Mestre, Ruth M. Risueño, Ana Manzanares, Pamela Acha, Blanca Xicoy and Francesc Solé Creator: LaTeX with hyperref Producer: pdfTeX-1.40.21 CreationDate: Tue Dec 5 14:44:53 2023 CET ModDate: Tue Dec 5 14:55:22 2023 CET Tagged: no UserProperties: no Suspects: no Form: none JavaScript: no Pages: 10 Encrypted: no Page size: 595.276 x 841.89 pts (A4) Page rot: 0 File size: 1349126 bytes Optimized: no PDF version: 1.7 name type encoding emb sub uni object ID ------------------------------------ ----------------- ---------------- --- --- --- --------- BTFGED+VnURWPalladioL-Bold Type 1 Custom yes yes no 10 0 QXDPCQ+VnURWPalladioL Type 1 Custom yes yes no 15 0 IKNYGL+URWPalladioL-Bold Type 1 Custom yes yes no 19 0 CVJUOP+URWPalladioL-Roma Type 1 Custom yes yes no 24 0 IHMSSU+URWPalladioL-Ital Type 1 Custom yes yes no 28 0 KWKPAT+CMSY10 Type 1 Builtin yes yes no 32 0 EBFBTV+TimesNewRomanPS-BoldItalicMT Type 1C WinAnsi yes yes no 50 0 CIDFont+F3 CID TrueType Identity-H yes no yes 77 0 CIDFont+F1 CID TrueType Identity-H yes no yes 85 0 CIDFont+F2 CID TrueType Identity-H yes no yes 93 0 OVNEXW+EURM10 Type 1 Builtin yes yes no 114 0 VPSNSA+VnURWPalladioL-Italic Type 1 Custom yes yes no 136 0 Jhove (Rel. 1.28.0, 2023-05-18) Date: 2024-02-21 02:38:29 CET RepresentationInformation: biomedicines-11-03222.pdf ReportingModule: PDF-hul, Rel. 1.12.4 (2023-03-16) LastModified: 2024-02-20 17:39:15 CET Size: 1349126 Format: PDF Version: 1.7 Status: Well-Formed and valid SignatureMatches: PDF-hul MIMEtype: application/pdf PDFMetadata: Objects: 238 FreeObjects: 1 IncrementalUpdates: 0 DocumentCatalog: PageLayout: SinglePage PageMode: UseNone Outlines: Item: Title: Introduction Destination: section.1 Item: Title: Materials and Methods Destination: section.2 Item: Title: Results Destination: section.3 Item: Title: Discussion Destination: section.4 Item: Title: References Destination: section.5 Info: Title: Two-Time Multiplexed Targeted Next-Generation Sequencing Might Help the Implementation of Germline Screening Tools for Myelodysplastic Syndromes/Hematologic Neoplasms Author: Oriol Calvete, Julia Mestre, Ruth M. Risueño, Ana Manzanares, Pamela Acha, Blanca Xicoy and Francesc Solé Subject: Next-generation sequencing (NGS) tools have importantly helped the classification of myelodysplastic syndromes (MDS), guiding the management of patients. However, new concerns are under debate regarding their implementation in routine clinical practice for the identification of germline predisposition. Cost-effective targeted NGS tools would improve the current standardized studies and genetic counseling. Here, we present our experience in a preliminary study detecting variants using a two-time multiplexed library strategy. Samples from different MDS patients were first mixed before library preparation and later multiplexed for a sequencing run. Two different mixes including a pool of three (3) and four (4) samples were evaluated. The filtered variants found in the individually sequenced samples were compared with the variants found in the two-time multiplexed studies to determine the detection efficiency scores. The same candidate variants were found in the two-time multiplexed studies in comparison with the individual tNGS. The variant allele frequency (VAF) values of the candidate variants were also compared. No significant differences were found between the expected and observed VAF percentages in both the 3 (p-value 0.74) and 4 (p-value 0.34) multiplexed studies. Our preliminary results suggest that the two-time multiplexing strategy might have the potential to help reduce the cost of evaluating germline predisposition. Keywords: myelodysplastic syndromes (MDS); genetic predisposition; targeted next-generation sequencing (tNGS); germline variants; genomic screening; multiplexed samples Creator: LaTeX with hyperref Producer: pdfTeX-1.40.21 CreationDate: Tue Dec 05 14:44:53 CET 2023 ModDate: Tue Dec 05 14:55:22 CET 2023 ID: 0xca1065ea2f82dd124df1b3d572256609, 0xca1065ea2f82dd124df1b3d572256609 Filters: FilterPipeline: FlateDecode Fonts: Type1: Font: BaseFont: KWKPAT+CMSY10 FontSubset: true FirstChar: 0 LastChar: 21 FontDescriptor: FontName: KWKPAT+CMSY10 Flags: Symbolic FontBBox: -29, -960, 1116, 775 FontFile: true Font: BaseFont: OVNEXW+EURM10 FontSubset: true FirstChar: 22 LastChar: 22 FontDescriptor: FontName: OVNEXW+EURM10 Flags: Symbolic FontBBox: -32, -243, 1060, 720 FontFile: true Font: BaseFont: IKNYGL+URWPalladioL-Bold FontSubset: true FirstChar: 3 LastChar: 241 FontDescriptor: FontName: IKNYGL+URWPalladioL-Bold Flags: Symbolic FontBBox: -152, -301, 1000, 935 FontFile: true EncodingDictionary: Differences: true Font: BaseFont: CVJUOP+URWPalladioL-Roma FontSubset: true FirstChar: 2 LastChar: 241 FontDescriptor: FontName: CVJUOP+URWPalladioL-Roma Flags: Symbolic FontBBox: -166, -283, 1021, 943 FontFile: true EncodingDictionary: Differences: true Font: BaseFont: VPSNSA+VnURWPalladioL-Italic FontSubset: true FirstChar: 178 LastChar: 226 FontDescriptor: FontName: VPSNSA+VnURWPalladioL-Italic Flags: Symbolic FontBBox: -170, -305, 1010, 941 FontFile: true EncodingDictionary: Differences: true Font: BaseFont: BTFGED+VnURWPalladioL-Bold FontSubset: true FirstChar: 178 LastChar: 178 FontDescriptor: FontName: BTFGED+VnURWPalladioL-Bold Flags: Symbolic FontBBox: -152, -301, 1000, 935 FontFile: true EncodingDictionary: Differences: true Font: BaseFont: IHMSSU+URWPalladioL-Ital FontSubset: true FirstChar: 46 LastChar: 121 FontDescriptor: FontName: IHMSSU+URWPalladioL-Ital Flags: Symbolic FontBBox: -170, -305, 1010, 941 FontFile: true EncodingDictionary: Differences: true Font: BaseFont: QXDPCQ+VnURWPalladioL FontSubset: true FirstChar: 161 LastChar: 254 FontDescriptor: FontName: QXDPCQ+VnURWPalladioL Flags: Symbolic FontBBox: -166, -283, 1021, 943 FontFile: true EncodingDictionary: Differences: true Pages: Page: Sequence: 1 Annotations: Annotation: Subtype: Link Rect: 121, 279, 152, 288 Annotation: Subtype: Link Rect: 34, 267, 150, 276 Annotation: Subtype: Link Rect: 34, 164, 96, 186 Annotation: Subtype: Link Rect: 126, 90, 154, 99 Annotation: Subtype: Link Rect: 34, 78, 147, 87 Annotation: Subtype: Link Rect: 34, 757, 197, 793 Annotation: Subtype: Link Rect: 515, 757, 560, 793 Annotation: Subtype: Link Rect: 109, 642, 120, 656 Annotation: Subtype: Link Rect: 192, 642, 203, 656 Annotation: Subtype: Link Rect: 297, 642, 308, 656 Annotation: Subtype: Link Rect: 395, 642, 406, 656 Annotation: Subtype: Link Rect: 475, 642, 486, 656 Annotation: Subtype: Link Rect: 129, 632, 140, 643 Annotation: Subtype: Link Rect: 549, 193, 556, 202 ActionDest: 130 Annotation: Subtype: Link Rect: 270, 68, 277, 76 ActionDest: 130 Annotation: Subtype: Link Rect: 473, 67, 480, 76 ActionDest: 130 Annotation: Subtype: Link Rect: 130, 17, 302, 28 Annotation: Subtype: Link Rect: 389, 17, 560, 28 Annotation: Subtype: Link Rect: 34, 388, 87, 407 Page: Sequence: 2 Annotations: Annotation: Subtype: Link Rect: 219, 673, 226, 682 ActionDest: 130 Annotation: Subtype: Link Rect: 226, 673, 233, 682 ActionDest: 130 Annotation: Subtype: Link Rect: 290, 660, 297, 669 ActionDest: 130 Annotation: Subtype: Link Rect: 461, 635, 468, 644 ActionDest: 130 Annotation: Subtype: Link Rect: 357, 610, 364, 619 ActionDest: 133 Annotation: Subtype: Link Rect: 333, 597, 340, 606 ActionDest: 133 Annotation: Subtype: Link Rect: 264, 572, 275, 581 ActionDest: 133 Annotation: Subtype: Link Rect: 253, 560, 265, 569 ActionDest: 133 Annotation: Subtype: Link Rect: 201, 409, 213, 418 ActionDest: 133 Annotation: Subtype: Link Rect: 216, 409, 228, 418 ActionDest: 133 Annotation: Subtype: Link Rect: 388, 396, 400, 406 ActionDest: 133 Annotation: Subtype: Link Rect: 415, 346, 427, 355 ActionDest: 133 Annotation: Subtype: Link Rect: 430, 346, 442, 355 ActionDest: 133 Annotation: Subtype: Link Rect: 365, 218, 372, 230 ActionDest: 72 Page: Sequence: 3 Annotations: Annotation: Subtype: Link Rect: 197, 236, 209, 245 ActionDest: 133 Annotation: Subtype: Link Rect: 326, 158, 333, 170 ActionDest: 72 Page: Sequence: 4 Annotations: Annotation: Subtype: Link Rect: 342, 683, 349, 695 ActionDest: 72 Annotation: Subtype: Link Rect: 194, 382, 201, 393 ActionDest: 111 Annotation: Subtype: Link Rect: 194, 139, 201, 151 ActionDest: 111 Page: Sequence: 5 Annotations: Annotation: Subtype: Link Rect: 438, 695, 445, 707 ActionDest: 118 Annotation: Subtype: Link Rect: 353, 404, 360, 416 ActionDest: 124 Annotation: Subtype: Link Rect: 448, 354, 455, 366 ActionDest: 124 Annotation: Subtype: Link Rect: 549, 266, 556, 278 ActionDest: 124 Annotation: Subtype: Link Rect: 549, 191, 556, 203 ActionDest: 124 Page: Sequence: 6 Page: Sequence: 7 Page: Sequence: 8 Annotations: Annotation: Subtype: Link Rect: 237, 565, 249, 574 ActionDest: 133 Annotation: Subtype: Link Rect: 433, 552, 445, 561 ActionDest: 133 Annotation: Subtype: Link Rect: 333, 527, 345, 536 ActionDest: 133 Annotation: Subtype: Link Rect: 478, 527, 490, 536 ActionDest: 133 Annotation: Subtype: Link Rect: 543, 514, 555, 523 ActionDest: 133 Annotation: Subtype: Link Rect: 300, 489, 312, 498 ActionDest: 133 Annotation: Subtype: Link Rect: 544, 477, 556, 486 ActionDest: 133 Annotation: Subtype: Link Rect: 456, 276, 468, 285 ActionDest: 133 Annotation: Subtype: Link Rect: 382, 125, 394, 134 ActionDest: 133 Annotation: Subtype: Link Rect: 401, 100, 413, 109 ActionDest: 133 Page: Sequence: 9 Annotations: Annotation: Subtype: Link Rect: 531, 725, 543, 734 ActionDest: 133 Annotation: Subtype: Link Rect: 289, 212, 327, 223 Annotation: Subtype: Link Rect: 333, 212, 369, 223 Annotation: Subtype: Link Rect: 397, 189, 434, 200 Annotation: Subtype: Link Rect: 441, 189, 477, 200 Annotation: Subtype: Link Rect: 126, 156, 164, 166 Annotation: Subtype: Link Rect: 100, 133, 137, 143 Annotation: Subtype: Link Rect: 143, 133, 179, 143 Annotation: Subtype: Link Rect: 118, 110, 155, 120 Annotation: Subtype: Link Rect: 161, 110, 197, 120 Annotation: Subtype: Link Rect: 443, 86, 481, 97 Annotation: Subtype: Link Rect: 487, 86, 523, 97 Annotation: Subtype: Link Rect: 59, 64, 96, 74 Page: Sequence: 10 Annotations: Annotation: Subtype: Link Rect: 59, 724, 96, 735 Annotation: Subtype: Link Rect: 311, 701, 348, 712 Annotation: Subtype: Link Rect: 104, 667, 142, 677 Annotation: Subtype: Link Rect: 131, 644, 168, 654 Annotation: Subtype: Link Rect: 175, 644, 211, 654 Annotation: Subtype: Link Rect: 59, 621, 96, 631 Annotation: Subtype: Link Rect: 402, 585, 439, 596 Annotation: Subtype: Link Rect: 445, 585, 481, 596 Annotation: Subtype: Link Rect: 404, 562, 441, 573 Annotation: Subtype: Link Rect: 447, 562, 483, 573 Annotation: Subtype: Link Rect: 455, 539, 492, 550 Annotation: Subtype: Link Rect: 104, 506, 142, 516 Annotation: Subtype: Link Rect: 148, 506, 184, 516 Annotation: Subtype: Link Rect: 176, 471, 214, 481 Annotation: Subtype: Link Rect: 488, 447, 525, 458 Annotation: Subtype: Link Rect: 313, 413, 350, 424 Annotation: Subtype: Link Rect: 517, 390, 554, 401 Annotation: Subtype: Link Rect: 275, 356, 313, 366 Annotation: Subtype: Link Rect: 319, 356, 355, 366 Annotation: Subtype: Link Rect: 59, 333, 96, 343 Annotation: Subtype: Link Rect: 102, 333, 138, 343 Annotation: Subtype: Link Rect: 59, 310, 96, 320 Annotation: Subtype: Link Rect: 102, 310, 138, 320 Annotation: Subtype: Link Rect: 144, 241, 182, 251 Annotation: Subtype: Link Rect: 225, 218, 263, 228 Checksum: d428afb5 Type: CRC32 Checksum: cfdfde2c64658b7d9626072dbb933870 Type: MD5 Checksum: 6aa274e8fedc457a71fb3095d4d71883a0a69fc3 Type: SHA-1 Checksum: eb56625903e5e681c3955e473ca3ba2727fdd92f69b6626b95273a718dc1b041 Type: SHA-256