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Applicability of next generation sequencing technology in microsatellite instability testing.

Gan C, Love C, Beshay V, Macrae F, Fox S, Waring P, Taylor G - Genes (Basel) (2015)

Bottom Line: Here, we describe a next generation sequencing approach for MSI testing using the MiSeq platform.Different from other MSI capturing strategies that are based on targeted gene capture, we utilize "deep resequencing", where we focus the sequencing on only the microsatellite regions of interest.Although there was some variation within individual markers, this NGS method produced the same overall MSI status for each tumour, as obtained with the traditional multiplex PCR-based method.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, Faculty of Medicine, Dentistry and Health Sciences, University of Melbourne, Parkville, Victoria 3052, Australia. c.gan3@student.unimelb.edu.au.

ABSTRACT
Microsatellite instability (MSI) is a useful marker for risk assessment, prediction of chemotherapy responsiveness and prognosis in patients with colorectal cancer. Here, we describe a next generation sequencing approach for MSI testing using the MiSeq platform. Different from other MSI capturing strategies that are based on targeted gene capture, we utilize "deep resequencing", where we focus the sequencing on only the microsatellite regions of interest. We sequenced a series of 44 colorectal tumours with normal controls for five MSI loci (BAT25, BAT26, BAT34c4, D18S55, D5S346) and a second series of six colorectal tumours (no control) with two mononucleotide loci (BAT25, BAT26). In the first series, we were able to determine 17 MSI-High, 1 MSI-Low and 26 microsatellite stable (MSS) tumours. In the second series, there were three MSI-High and three MSS tumours. Although there was some variation within individual markers, this NGS method produced the same overall MSI status for each tumour, as obtained with the traditional multiplex PCR-based method.

No MeSH data available.


Related in: MedlinePlus

Data derived from NGS-MSI-High tumour. The upper and lower panels correspond to three mononucleotide loci and two dinucleotide loci, respectively. The tumour and normal tissue are represented by the black and grey columns, respectively. There is a deletion in the base pair length (3–6 base pairs) for each mononucleotide locus in the tumour compared to normal tissue. D18S55 demonstrates an allelic loss and deletion (8 base pairs) in the base pair length. D5S346 shows expansion and deletion (4–8 base pairs) in the base pair length.
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genes-06-00046-f002: Data derived from NGS-MSI-High tumour. The upper and lower panels correspond to three mononucleotide loci and two dinucleotide loci, respectively. The tumour and normal tissue are represented by the black and grey columns, respectively. There is a deletion in the base pair length (3–6 base pairs) for each mononucleotide locus in the tumour compared to normal tissue. D18S55 demonstrates an allelic loss and deletion (8 base pairs) in the base pair length. D5S346 shows expansion and deletion (4–8 base pairs) in the base pair length.

Mentions: Using our protocol as described in the Materials and Methods, we determined 17 MSI-H, 26 MSS and one MSI-L tumours (Table 3). The overall MSI results for all tumours were 100% concordant with the multiplex PCR-based method (data not shown [20,21]) only when we grouped MSS and MSI-L tumours together. In Case 21, the D18S55 locus was unstable by NGS (MSI loci for the multiplex PCR-based method were all stable; D18S55 was not included in the multiplex PCR panel), resulting in a classification of MSI-L rather than MSS. Examples of MSI-H and MSS tumours are shown in Figure 2 and Figure 3.


Applicability of next generation sequencing technology in microsatellite instability testing.

Gan C, Love C, Beshay V, Macrae F, Fox S, Waring P, Taylor G - Genes (Basel) (2015)

Data derived from NGS-MSI-High tumour. The upper and lower panels correspond to three mononucleotide loci and two dinucleotide loci, respectively. The tumour and normal tissue are represented by the black and grey columns, respectively. There is a deletion in the base pair length (3–6 base pairs) for each mononucleotide locus in the tumour compared to normal tissue. D18S55 demonstrates an allelic loss and deletion (8 base pairs) in the base pair length. D5S346 shows expansion and deletion (4–8 base pairs) in the base pair length.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4377833&req=5

genes-06-00046-f002: Data derived from NGS-MSI-High tumour. The upper and lower panels correspond to three mononucleotide loci and two dinucleotide loci, respectively. The tumour and normal tissue are represented by the black and grey columns, respectively. There is a deletion in the base pair length (3–6 base pairs) for each mononucleotide locus in the tumour compared to normal tissue. D18S55 demonstrates an allelic loss and deletion (8 base pairs) in the base pair length. D5S346 shows expansion and deletion (4–8 base pairs) in the base pair length.
Mentions: Using our protocol as described in the Materials and Methods, we determined 17 MSI-H, 26 MSS and one MSI-L tumours (Table 3). The overall MSI results for all tumours were 100% concordant with the multiplex PCR-based method (data not shown [20,21]) only when we grouped MSS and MSI-L tumours together. In Case 21, the D18S55 locus was unstable by NGS (MSI loci for the multiplex PCR-based method were all stable; D18S55 was not included in the multiplex PCR panel), resulting in a classification of MSI-L rather than MSS. Examples of MSI-H and MSS tumours are shown in Figure 2 and Figure 3.

Bottom Line: Here, we describe a next generation sequencing approach for MSI testing using the MiSeq platform.Different from other MSI capturing strategies that are based on targeted gene capture, we utilize "deep resequencing", where we focus the sequencing on only the microsatellite regions of interest.Although there was some variation within individual markers, this NGS method produced the same overall MSI status for each tumour, as obtained with the traditional multiplex PCR-based method.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, Faculty of Medicine, Dentistry and Health Sciences, University of Melbourne, Parkville, Victoria 3052, Australia. c.gan3@student.unimelb.edu.au.

ABSTRACT
Microsatellite instability (MSI) is a useful marker for risk assessment, prediction of chemotherapy responsiveness and prognosis in patients with colorectal cancer. Here, we describe a next generation sequencing approach for MSI testing using the MiSeq platform. Different from other MSI capturing strategies that are based on targeted gene capture, we utilize "deep resequencing", where we focus the sequencing on only the microsatellite regions of interest. We sequenced a series of 44 colorectal tumours with normal controls for five MSI loci (BAT25, BAT26, BAT34c4, D18S55, D5S346) and a second series of six colorectal tumours (no control) with two mononucleotide loci (BAT25, BAT26). In the first series, we were able to determine 17 MSI-High, 1 MSI-Low and 26 microsatellite stable (MSS) tumours. In the second series, there were three MSI-High and three MSS tumours. Although there was some variation within individual markers, this NGS method produced the same overall MSI status for each tumour, as obtained with the traditional multiplex PCR-based method.

No MeSH data available.


Related in: MedlinePlus