Determination of RET Sequence Variation in an MEN2 Unaffected Cohort Using Multiple-Sample Pooling and Next-Generation Sequencing

Table 1

Variants detected in the pooled data sets and their NGS percent variant read values^a.

RET amplicon position	Chr 10 positions	RET gene location	Genotype	dbSNP	dbSNP allele frequency	Found only in which pool	Ethnic	Caucasian combined pop freq	P1	P2	P3	P4	Control
							% variant reads		% variant reads	% variant reads	% variant reads	% variant reads	% variant reads
117	43,608,807	Intron 9	c.1760−197G>T	112675631	8.0%		6.20%	6.19%	5.62%	8.05%	7.77%	2.90%	0.00%
156	43,608,846	Intron 9	c.1760−158C>G	Novel		Caucasian	0.01%	0.88%	0.01%	1.12%	0.00%	1.49%	0.00%
174	43,608,864	Intron 9	c.1760−140C>G	3026758		Ethnic	6.66%	0.00%	0.00%	0.00%	0.00%	0.00%	0.00%
819	43,609,509	Intron 10	c.1880−419G>A	Novel		Caucasian	0.05%	0.44%	0.06%	1.85%	0.05%	0.05%	0.06%
1429	43,610,119	exon 11	c.2071G>A	1799939	14.7%		12.79%	18.14%	15.00%	15.33%	17.30%	24.78%	0.07%
1645	43,610,335	Intron 11	c.2136+151G>A	Novel		Caucasian	0.10%	0.44%	1.92%	0.10%	0.10%	0.10%	0.12%
1676	43,610,366	Intron 11	c.2136+182G>A	1864400	75.2%		68.62%	75.66%	79.53%	76.35%	68.79%	79.64%	0.02%
1765	43,610,455	Intron 11	c.2136+271T>C	1864399	73.2%		63.96%	69.47%	72.74%	67.72%	62.75%	75.90%	0.10%
1766	43,610,456	Intron 11	c.2136+272G>A	Novel		Caucasian	0.03%	0.44%	1.79%	0.04%	0.03%	0.03%	0.02%
1868	43,610,558	Intron 11	c.2136+374C>T	2742233	72.3%		61.79%	69.47%	73.34%	67.78%	61.11%	76.34%	0.07%
1931	43,610,621	Intron 11	c.2136+437T>C	Novel		Caucasian	0.16%	0.44%	0.15%	0.14%	2.15%	0.17%	0.13%
1981	43,610,671	Intron 11	c.2136+487G>T	3026762	6.2%		6.09%	9.29%	8.80%	3.32%	10.17%	13.42%	0.01%
2003	43,610,693	Intron 11	c.2136+509G>T	Novel		Caucasian	0.01%	0.44%	1.75%	0.00%	0.01%	0.01%	0.00%
2096	43,610,786	Intron 11	c.2136+602T>C	Novel		Caucasian	0.08%	0.88%	1.59%	1.61%	0.06%	0.07%	0.06%
2207	43,610,897	Intron 11	c.2136+713C>T	Novel		Caucasian	0.04%	0.44%	0.04%	1.84%	0.03%	0.03%	0.03%
2598	43,611,288	Intron 11	c.2137−744A>G	50 pool		Caucasian	0.23%	1.33%	1.94%	1.89%	0.22%	1.68%	0.24%
2618	43,611,308	Intron 11	c.2137−724G>A	Novel		Caucasian	0.07%	0.44%	1.92%	0.06%	0.07%	0.06%	0.05%
2958	43,611,648	Intron 11	c.2137−384C>T	50 pool			1.92%	0.44%	0.08%	1.69%	0.09%	0.09%	0.12%
3018	43,611,708	Intron 11	c.2137−324A>G	741968	76.0%		68.18%	76.11%	78.72%	76.37%	68.79%	81.20%	0.04%
3089	43,611,779	Intron 11	c.2137−253C>T	74135468	6.9%	Ethnic	1.54%	0.00%	0.03%	0.03%	0.04%	0.04%	0.04%
3175	43,611,865	Intron 11	c.2137−167T>C	2256550	47.2%		46.33%	45.58%	51.81%	45.88%	39.24%	45.15%	0.22%
3535	43,612,225	Intron 12	c.2284+46G>C	Novel		Ethnic	1.85%	0.00%	0.00%	0.00%	0.00%	0.01%	0.00%
3536	43,612,226	Intron 12	c.2284+47C>T	760466	14.5%		10.07%	17.26%	18.80%	15.39%	14.88%	20.45%	0.05%
3919	43,612,609	Intron 12	c.2284+430C>T	2742234	70.1%		62.23%	69.91%	73.95%	68.56%	60.98%	76.51%	0.04%
4382	43,613,072	Intron 12	c.2285−749C>T	3026765	2.2%	Caucasian	0.04%	4.87%	5.61%	6.82%	3.42%	3.62%	0.05%
4418	43,613,108	Intron 12	c.2285−713G>A	79045327	0.4%		1.97%	0.44%	0.06%	1.71%	0.06%	0.05%	0.07%
4503	43,613,193	Intron 12	c.2285−628T>C	Novel		Caucasian	0.23%	0.44%	1.99%	0.23%	0.23%	0.24%	0.25%
4710	43,613,400	Intron 12	c.2285−421G>A	114921735	2.9%	Ethnic	1.86%	0.00%	0.11%	0.08%	0.09%	0.08%	0.11%
4750	43,613,440	Intron 12	c.2285−381G>A	Novel		Ethnic	2.15%	0.00%	0.09%	0.08%	0.07%	0.09%	0.10%
5153	43,613,843	Exon 13	c.2307G>T	1800861	72.3%		62.24%	70.35%	74.10%	68.58%	61.27%	76.98%	0.01%
5397	43,614,087	Intron 13	c.2392+159G>A	3026767	1.6%	Caucasian	0.03%	3.54%	3.37%	1.71%	4.93%	3.68%	0.02%
5540	43,614,230	Intron 13	c.2392+302G>A	2075910	72.2%		62.45%	70.35%	74.57%	68.59%	61.28%	76.56%	0.04%
5630	43,614,320	Intron 13	c.2392+392G>A	Novel		Caucasian	0.04%	0.44%	0.03%	0.03%	0.02%	1.60%	0.03%
5752	43,614,442	Intron 13	c.2392+514G>A	50 pool		Caucasian	0.06%	0.88%	0.06%	1.76%	1.93%	0.06%	0.07%
5770	43,614,460	Intron 13	c.2393−519G>A	2075911	14.7%		12.43%	18.14%	14.55%	14.88%	17.04%	24.21%	0.04%
5820	43,614,510	Intron 13	c.2393−469C>A	Novel		Caucasian	0.02%	0.44%	1.82%	0.01%	0.01%	0.01%	0.01%
6004	43,614,694	Intron 13	c.2393−285G>A	78453984	5.4%	Caucasian	0.03%	0.44%	0.03%	0.02%	0.03%	1.52%	0.03%
6195	43,614,885	Intron 13	c.2393−94C>T	111264957	4.2%		5.77%	6.19%	5.22%	7.80%	7.37%	3.01%	0.09%
6221	43,614,911	Intron 13	c.2393−68A>G	Novel		Ethnic	2.19%	0.00%	0.18%	0.15%	0.15%	0.15%	0.12%
6404	43,615,094	exon 14	c.2508C>T	1800862	4.1%		5.86%	5.75%	5.13%	7.67%	7.39%	2.98%	0.04%
6639	43,615,329	Intron 14	c.2607+136A>G	50 pool			2.06%	0.44%	0.16%	1.90%	0.15%	0.17%	0.14%
6692	43,615,382	Intron 14	c.2608−147C>T	11238441	14.8%		12.78%	18.58%	14.55%	15.09%	17.30%	25.23%	0.06%
6696	43,615,386	Intron 14	c.2608−143C>G	Novel		Ethnic	1.90%	0.00%	0.00%	0.00%	0.00%	0.00%	0.00%
6715	43,615,405	Intron 14	c.2608−124G>A	111306965	8.9%	Ethnic	1.93%	0.00%	0.04%	0.06%	0.04%	0.05%	0.06%
6815	43,615,505	Intron 14	c.2608−24G>A	2472737	16.0%		19.84%	17.26%	21.52%	18.75%	14.39%	13.81%	0.04%
6943	43,615,633	Exon 15	c.2712C>G	1800863	14.9%		11.74%	16.37%	13.17%	13.92%	16.24%	22.34%	0.00%
7190	43,615,880	Intron 15	c.2730+229T>C	3026768	5.1%		6.07%	6.64%	5.40%	8.07%	7.75%	4.57%	0.21%
7218	43,615,908	Intron 15	c.2730+257C>T	2435353	14.4%		19.91%	17.26%	21.81%	18.89%	14.51%	14.19%	0.08%
7283	43,615,973	Intron 15	c.2730+322C>T	3026769	4.3%		1.79%	1.33%	0.07%	1.66%	1.68%	1.37%	0.06%
7392	43,616,082	Intron 15	c.2730+431G>A	Novel		Ethnic	1.93%	0.00%	0.17%	0.13%	0.17%	0.17%	0.18%
7491	43,616,181	Intron 15	c.2730+530A>G	79094522	11.1%	Caucasian	0.21%	0.44%	0.25%	0.23%	0.20%	1.66%	0.22%
7635	43,616,325	Intron 15	c.2730+674A>G	2742235	76.0%		68.78%	76.99%	79.66%	77.02%	71.18%	79.24%	0.18%
7844	43,616,534	Intron 15	c.2731−860G>A	Novel		Ethnic	1.94%	0.00%	0.02%	0.03%	0.02%	0.03%	0.02%
8061	43,616,751	Intron 15	c.2731−643C>A	715106	76.7%		68.41%	77.43%	79.60%	77.02%	70.90%	80.13%	0.01%
8179	43,616,869	Intron 15	c.2731−525G>A	Novel		Caucasian	0.04%	0.44%	1.80%	0.03%	0.03%	0.04%	0.03%
8181	43,616,871	Intron 15	c.2731−523T>G	Novel		Caucasian	0.01%	0.88%	0.00%	0.00%	0.01%	3.48%	0.00%
8328	43,617,018	Intron 15	c.2731−376A>G	3026770	1.0%	Caucasian	0.17%	0.88%	0.17%	0.17%	1.81%	1.47%	0.15%
8414	43,617,104	Intron 15	c.2731−290A>G	2565202	47.6%		47.17%	52.21%	58.83%	52.76%	45.68%	50.20%	0.05%
8477	43,617,167	Intron 15	c.2731−227C>G	3026771	4.5%		5.82%	6.19%	5.09%	7.64%	7.36%	4.31%	0.00%
8828	43,617,518	intron 16	c.2801+54A>T	3026772	0.5%	Caucasian	0.01%	0.44%	0.00%	0.00%	0.00%	1.66%	0.00%
9017	43,617,707	intron 16	c.2801+243G>C	3026774	2.1%	Caucasian	0.02%	5.75%	5.21%	6.66%	6.77%	3.63%	0.00%

^aTable headings. RET amplicon position and Chr 10: amplicon positions 1–9180 correlate to Chr 10 positions 43608691–43617870 in reference sequence NC_000010.10. Genotype: nomenclature (cDNA) for known variants is from the MEN2 RET database—http://www.arup.utah.edu/database/MEN2/MEN2_welcome.php, which uses the Human Genome Variation Society sequence variation nomenclature and RET reference sequence NC_000010.10. dbSNP column lists rs number, “50 pool” (if novel from dbSNP, but was found in 50 pool data set), or “novel” change. Found only in which pool: variation was found only within the ethnic or Caucasian pools. % variant reads: NGS determined variant read percentage for each variant found within each pool. Ethnic: data was from 23 pooled non-Caucasian samples. Caucasian combined pop freq: it combines all the data from the 4 Caucasian pools (P1, P2, P3, and P4), for comparison to the ethnic pool as well as the NCBI dbSNP 132 stated allele frequencies. Control: this single-sample control matched the reference sequence exactly, so no variations were detected and the values in this control column are only background sequencing error rates (0% to 0.25% variant read values).