Background: To support cancer therapy, development of low cost library preparation techniques for targeted next generation sequencing (NGS) is needed. In this study we designed and tested a PCR-based library preparation panel with limited target area for sequencing the top 12 somatic mutation hot spots in @entity14 on the GS Junior instrument. Materials and Methods: A multiplex PCR panel was designed to amplify regions of mutation hot spots in 12 selected genes ( @entity1412 , @entity817 , @entity3546 , @entity470 , @entity26562 , @entity984 , @entity8497 , @entity4638 , @entity10160 , @entity2536 , @entity1594 , @entity876 ). Amplicons were sequenced on a GS Junior instrument using ligated and barcoded adaptors. Eight samples were sequenced in a single run. Colonic DNA samples (8 normal mucosa; 33 @entity652 ; 17 @entity420 ) as well as HT-29 and Caco-2 cell lines with known mutation profiles were analyzed. Variants found by the panel on @entity1412 , @entity817 , @entity984 and @entity8497 genes were validated by conventional sequencing. Results: In total, 34 kinds of mutations were detected including two novel mutations ( @entity26562 c.1740:C>G and @entity1594 @entity69434 ) that have not been recorded in mutation databases, and one potential germline mutation ( @entity1412 ). The most frequently mutated genes were @entity1412 , @entity876 and @entity984 with 30%, 15% and 21% frequencies in @entity652 and 29%, 53% and 29% frequencies in @entity189 , respectively. In cell lines, all the expected mutations were detected except for one located in a homopolymer region. According to re-sequencing results sensitivity and specificity was 100% and 92% respectively. Conclusions: Our NGS-based screening panel denotes a promising step towards low cost @entity14 genotyping on the GS Junior instrument. Despite the relatively low coverage, we discovered two novel mutations and obtained mutation frequencies comparable to literature data. Additionally, as an advantage, this panel requires less template DNA than sequence capture @entity694 panels currently available for the GS Junior instrument.

Construction of a multiplex mutation hot spot PCR panel: the first step towards XXXX genotyping on the GS Junior platform.

@entity652 :: ('MESH:D000236', 'Disease') :: ['adenomas'] @entity470 :: ('1956', 'Gene') :: ['EGFR'] @entity984 :: ('3845', 'Gene') :: ['KRAS'] @entity2536 :: ('4087', 'Gene') :: ['SMAD2'] @entity189 :: ('MESH:D002277', 'Disease') :: ['carcinomas'] @entity69434 :: ('c|SUB|C|413|G', 'DNAMutation') :: ['c.413C>G'] @entity420 :: ('MESH:D000230', 'Disease') :: ['adenocarcinomas'] @entity3546 :: ('1499', 'Gene') :: ['CTNNB1'] @entity26562 :: ('55294', 'Gene') :: ['FBXW7'] @entity876 :: ('7157', 'Gene') :: ['TP53'] @entity817 :: ('673', 'Gene') :: ['BRAF'] @entity14 :: ('MESH:D015179', 'Disease') :: ['colorectal cancer'] @entity8497 :: ('4893', 'Gene') :: ['NRAS'] @entity1594 :: ('4089', 'Gene') :: ['SMAD4'] @entity1412 :: ('MESH:D011125', 'Disease') :: ['APC'] @entity694 :: ('MESH:D003110', 'Disease') :: ['colon cancer'] @entity10160 :: ('5290', 'Gene') :: ['PIK3CA'] @entity4638 :: ('2956', 'Gene') :: ['MSH6']
@entity14 :: (MESH:D015179,Disease) :: ['colorectal cancer']
