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To aid clinical laboratories in testing the most clinically significant genes, along with the published ACMG CRC technical standard guidelines, this protocol aims to provide a step\u2010by\u2010step technical workflow for carrying out the NGS\u2010panel based CRC molecular diagnosis focusing on the wet lab portion of library preparation and massively parallel sequencing. Using the most popular pull\u2010down\u2010based target enrichment, the chapter particularly encompasses genomic DNA (gDNA) fragmentation, adapter ligation, indexing, hybridization, and capture, which is the most variable and technically challenging part of NGS testing involving at least 3 quality control (QC) checkpoints plus the pre\u2010 and post\u2010capture PCR. The gDNA extraction and sequencing is less covered because they are relatively standard technologies with little variations and choices. Although this protocol also introduces pertinent testing algorithms and a brief guideline for pre\u2010 and post\u2010testing genetic counselling, the audiences are required to refer to National Comprehensive Cancer Network (NCCN) clinical practice guidelines to determine the most appropriate testing strategies. Since NGS panel\u2010based testing is a highly complex and dynamic platform with multiple choices from different technology and commercial resources, this technical benchtop\u2010based protocol also aims to cover some of the key ramification points for decision\u2010making by each laboratory at the discretion of the directors. \u00a9 2023 Wiley Periodicals LLC.<\/jats:p><jats:p><jats:bold>Basic Protocol<\/jats:bold>: Hereditary colorectal cancer (CRC) diagnosis by next\u2010generation sequencing<\/jats:p>","DOI":"10.1002\/cpz1.941","type":"journal-article","created":{"date-parts":[[2023,12,19]],"date-time":"2023-12-19T14:26:06Z","timestamp":1702995966000},"source":"Crossref","is-referenced-by-count":1,"title":["Hereditary Colorectal Cancer Diagnosis by Next\u2010Generation Sequencing"],"prefix":"10.1002","volume":"3","author":[{"ORCID":"https:\/\/orcid.org\/0009-0006-7209-5565","authenticated-orcid":false,"given":"Ting","family":"Wen","sequence":"first","affiliation":[{"name":"University of Utah School of Medicine  Salt Lake City Utah"},{"name":"ARUP Laboratories  Salt Lake City Utah"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Fabienne","family":"Ehivet","sequence":"additional","affiliation":[{"name":"Department of Human Genetics Emory University School of Medicine  Atlanta Georgia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Christine","family":"Stanislaw","sequence":"additional","affiliation":[{"name":"Department of Human Genetics Emory University School of Medicine  Atlanta Georgia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Rong","family":"Mao","sequence":"additional","affiliation":[{"name":"University of Utah School of Medicine  Salt Lake City Utah"},{"name":"ARUP Laboratories  Salt Lake City Utah"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Madhuri","family":"Hegde","sequence":"additional","affiliation":[{"name":"Department of Human Genetics Emory University School of Medicine  Atlanta Georgia"},{"name":"Revvity  Waltham Massachusetts"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"311","published-online":{"date-parts":[[2023,12,19]]},"reference":[{"key":"e_1_2_6_2_1_1","doi-asserted-by":"publisher","DOI":"10.1056\/NEJMoa2035790"},{"volume-title":"Colorectal Cancer Risk Factors 2020\u20102022","year":"2020","author":"American Cancer Society","key":"e_1_2_6_2_2_1"},{"key":"e_1_2_6_2_3_1","doi-asserted-by":"publisher","DOI":"10.1200\/JCO.2003.03.189"},{"key":"e_1_2_6_2_4_1","doi-asserted-by":"publisher","DOI":"10.1038\/s42003\u2010022\u201003102\u20108"},{"volume-title":"Understanding Genetics: A New England Guide for Patients and Health Professionals","year":"2010","author":"Genetic Alliance","key":"e_1_2_6_2_5_1"},{"key":"e_1_2_6_2_6_1","doi-asserted-by":"publisher","DOI":"10.1007\/s10350\u2010004\u20106397\u20104"},{"key":"e_1_2_6_2_7_1","doi-asserted-by":"publisher","DOI":"10.1007\/s10689\u2010004\u20100899\u2010z"},{"key":"e_1_2_6_2_8_1","doi-asserted-by":"publisher","DOI":"10.1002\/jgc4.1546"},{"key":"e_1_2_6_2_9_1","doi-asserted-by":"publisher","DOI":"10.1136\/jmg.33.8.636"},{"volume-title":"Gene Reviews [Internet]","year":"2004","author":"Idos G.","key":"e_1_2_6_2_10_1"},{"key":"e_1_2_6_2_11_1","doi-asserted-by":"publisher","DOI":"10.1007\/s10689\u2010011\u20109507\u20101"},{"key":"e_1_2_6_2_12_1","doi-asserted-by":"publisher","DOI":"10.1038\/s41436\u2010021\u201001187\u2010w"},{"key":"e_1_2_6_2_13_1","doi-asserted-by":"publisher","DOI":"10.1001\/jama.291.6.718"},{"key":"e_1_2_6_2_14_1","doi-asserted-by":"publisher","DOI":"10.1038\/s41436\u2010021\u201001207\u20109"},{"volume-title":"StatPearls [Internet]","year":"2022","author":"Medina Pab\u00f3n M. 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