酶切法建庫試劑盒，選擇翌圣OnePot
翌圣OnePot系列是針對(duì)Illumina®測(cè)序平臺(tái)定向研發(fā)的酶切法DNA建庫試劑盒，采用高質(zhì)量片段化酶，擺脫了繁瑣的超聲過程，同時(shí)簡(jiǎn)化了操作流程，極大的降低了建庫的時(shí)間和成本。具有優(yōu)秀的文庫轉(zhuǎn)化率與文庫擴(kuò)增效率。
隨著NGS技術(shù)的高速發(fā)展，人們對(duì)于效率的要求越來越高，在消耗同等的時(shí)間和精力下，人們更愿意選擇快速，高效的方法。
超聲打斷（機(jī)械法）是NGS建庫中常用的DNA片段化方法之一，由于該方法過度依賴超聲儀器和搭配的實(shí)驗(yàn)耗材，再加上耗時(shí)較長(zhǎng)，被越來越多的客戶所放棄。
翌圣OnePot系列酶切法DNA建庫試劑盒，擺脫了繁瑣的超聲流程，操作簡(jiǎn)便化，針對(duì)不同樣本(含F(xiàn)FPE)均可以獲得高質(zhì)量文庫，是自動(dòng)化平臺(tái)建庫的首選。
為什么選擇OnePot代替機(jī)械法建庫
一、 精簡(jiǎn)流程
合并建庫方法，優(yōu)化建庫流程，手工操作低于0.5h，整體文庫構(gòu)建僅需2h，真正的高效建庫。
二、 可控酶切
酶切產(chǎn)物片段大小只與酶切時(shí)間有關(guān)，簡(jiǎn)便可控，適用于個(gè)性化建庫和自動(dòng)化建庫。
三、 兼容FFPE高效建庫
1） 適用于不同程度降解的FFPE樣本
對(duì)應(yīng)上述樣本建庫詳情
|
編號(hào) |
1 |
2 |
3 |
4 |
5 |
6 |
7 |
8 |
9 |
10 |
11 |
|
Input DNA（ng） |
50 |
50 |
50 |
50 |
50 |
50 |
50 |
50 |
50 |
10 |
10 |
|
cycles |
8 |
8 |
8 |
8 |
8 |
8 |
8 |
8 |
8 |
10 |
10 |
|
酶切時(shí)間 （min） |
6 |
6 |
6 |
6 |
6 |
6 |
6 |
6 |
6 |
6 |
10 |
|
濃度（ng/ul） 32ul洗脫 |
35.6 |
63.8 |
23.2 |
33.8 |
48.4 |
53.6 |
165.6 |
136.8 |
104 |
117.6 |
136 |
|
Total (μg) |
1.14 |
2.04 |
0.74 |
1.08 |
1.55 |
1.72 |
5.3 |
4.38 |
3.33 |
3.76 |
4.35 |
2）低質(zhì)量FFPE樣本，搭配修復(fù)試劑建庫效果更佳
四、 適用范圍廣
適用于不同質(zhì)量的痰液，腹水等微量建庫樣本
|
樣本編號(hào) |
642-痰液 |
640-痰液 |
666-腹水 |
637-腹水 |
642-痰液 |
640-痰液 |
666-腹水 |
637-腹水 |
|
濃度（ng/μl） |
16.3 |
53.0 |
24.6 |
8.16 |
16.3 |
53.0 |
24.6 |
8.16 |
|
Input DNA |
10 ng |
10 ng |
10 ng |
10 ng |
10 ng |
10 ng |
10 ng |
10 ng |
|
酶切時(shí)間（min） |
13 |
10 |
||||||
|
Cycles |
9 |
9 |
9 |
9 |
9 |
9 |
9 |
9 |
|
產(chǎn)量（μg） |
2.58 |
1.32 |
2.44 |
1.39 |
2.21 |
0.91 |
2.26 |
1.21 |
五、 精準(zhǔn)的數(shù)據(jù)檢出率
1） 標(biāo)準(zhǔn)品Horizon@HD753的檢出率與實(shí)際基本無差別
|
Variant Type |
Chromosome Number |
Gene |
Variant |
Expected Allelic Frequency, % |
Chr |
position |
nt D |
102panel 1:2 |
|
SNV High GC |
chr.19 |
GNA11 |
Q209L |
5.6 |
19 |
3118942 |
A>T |
6.13% |
|
SNV High GC |
chr.14 |
AKT1 |
E17K |
5 |
14 |
105246551 |
G>A |
4.78% |
|
SNV Low GC |
chr.3 |
PIK3CA |
E545K |
5.6 |
3 |
178936091 |
G>A |
5.23% |
|
Long Insertion |
chr.7 |
EGFR |
V769_D770insASV |
5.6 |
7 |
Exon 20 insertion |
GG>GGCCAGCGTGG |
4.54% |
|
Long Deletion |
chr.7 |
EGFR |
ΔE746 - A750 |
5.3 |
7 |
55242465-55242479 |
AGGAATTAAGAGAAGC>A |
3.52% |
|
Fusion |
chr.4:chr.6 |
ROS1 |
SLC34A2/ROS1 fusion |
5.6 |
4-6 |
fusing exon 2 of SLC34A2 to exon 32 or 34 of ROS1 |
CTGGXTCTGGAGATCTGGCTGGAGTCCCAAATAA,、 CTGGXTCTGGAGATCTGG ATGATTTTTGGATACCA |
3.58% |
|
Fusion |
chr.10 |
RET |
CCDC6/RET fusion |
5 |
10-10 |
CCDC6 (EXON1) /RET (EXON12) |
TGCGCAAAGCCAGCGTTACCATCGAGGATCCAAAGTGGGAATTC |
5.86% |
|
CNV |
chr.7 |
MET |
amplification |
4.5 copies |
7 |
116312459-116340338 |
4.4 |
|
|
CNV |
chr.2 |
MYC-N |
amplification |
9.5 copies |
2 |
16080560-16087129 |
||
|
期望值 |
實(shí)際檢測(cè)值 |
2）FFPE DNA標(biāo)準(zhǔn)品（Horizon HD200）
捕獲建庫，IGT 944K cancer panel T074V1
OnePot檢測(cè)到突變率的準(zhǔn)確性與Enzymatics相當(dāng)（*紅色標(biāo)注為更接近預(yù)期突變）
|
clean |
all_map_reads |
map ratio (%) |
Target effective reads |
reads capture rate (%) |
Dup (%) |
Coverage (%) |
20%X depth cov (%) |
|
|
OnePot (with MaxUp II amplification mix) |
17716013 |
17674611 |
99.7663018 |
15721075 |
88.94721926 |
31.8432 |
99.9895144 |
99.48491028 |
|
Enzymatics (with KAPA HiFi Readymix) |
13805070 |
13775426 |
99.7852673 |
8147070 |
59.14205484 |
25.9288 |
99.9739449 |
99.68490276 |
|
Chromosome |
Gene |
Variant |
Expected Allelic Frequency, % |
Original base |
Mutant base |
Frequency/OnePot,% |
Frequency/Enzymatics,% |
|
7q34 |
BRAF |
V600E |
10.70% |
A |
T |
11.7180617 |
10.0371747 |
|
4q11-q12 |
cKIT |
D816V |
10.00% |
A |
T |
9.798995 |
25.2446184 |
|
7p12 |
EGFR |
ΔE746 - A750 |
1.90% |
GGAATTAAGAGAAGC |
- |
0.9146341 |
1.1053541 |
|
7p12 |
EGFR |
L858R |
2.80% |
T |
G |
3.2357907 |
3.6181678 |
|
7p12 |
EGFR |
T790M |
0.90% |
C |
T |
1.0137989 |
1.6100179 |
|
7p12 |
EGFR |
G719S |
24.50% |
G |
A |
21.8106996 |
21.4709913 |
|
12p12.1 |
KRAS |
G13D |
15.00% |
C |
T |
11.6261398 |
10.6679035 |
|
12p12.1 |
KRAS |
G12D |
6.30% |
C |
T |
3.245283 |
4.0613718 |
|
1p13.2 |
NRAS |
Q61K |
12.50% |
G |
T |
10.7090465 |
12.1263395 |
|
3q26.3 |
PI3KCA |
H1047R |
17.50% |
A |
G |
14.9758454 |
15.7051282 |
|
3q26.3 |
PI3KCA |
E545K |
8.80% |
G |
A |
9.798995 |
15.6359393 |
市場(chǎng)使用反饋
一、 上海某生物公司X*
實(shí)驗(yàn)樣本：不同質(zhì)量的FFPE樣本
樣本數(shù)目：4（兩個(gè)質(zhì)量較好，兩個(gè)質(zhì)量較差）
實(shí)驗(yàn)結(jié)論：針對(duì)客戶提供的2個(gè)質(zhì)量較好樣本（樣本3/4），建庫產(chǎn)量可遠(yuǎn)高于其預(yù)期值，對(duì)于質(zhì)量較差樣本（樣本1/2）,使用12606（H5901150）FFPE樣本修復(fù)試劑可明顯改善其產(chǎn)量，產(chǎn)量均可達(dá)客戶需求。建議客戶針對(duì)質(zhì)量較差FFPE樣本使用我公司12606產(chǎn)品進(jìn)行修復(fù)后再行建庫以獲得更高產(chǎn)量。
|
Chromosome |
Gene |
Variant |
Expected Allelic Frequency, % |
Original base |
Mutant base |
Frequency/OnePot,% |
Frequency/Enzymatics,% |
|
7q34 |
BRAF |
V600E |
10.70% |
A |
T |
11.7180617 |
10.0371747 |
|
4q11-q12 |
cKIT |
D816V |
10.00% |
A |
T |
9.798995 |
25.2446184 |
|
7p12 |
EGFR |
ΔE746 - A750 |
1.90% |
GGAATTAAGAGAAGC |
- |
0.9146341 |
1.1053541 |
|
7p12 |
EGFR |
L858R |
2.80% |
T |
G |
3.2357907 |
3.6181678 |
|
7p12 |
EGFR |
T790M |
0.90% |
C |
T |
1.0137989 |
1.6100179 |
|
7p12 |
EGFR |
G719S |
24.50% |
G |
A |
21.8106996 |
21.4709913 |
|
12p12.1 |
KRAS |
G13D |
15.00% |
C |
T |
11.6261398 |
10.6679035 |
|
12p12.1 |
KRAS |
G12D |
6.30% |
C |
T |
3.245283 |
4.0613718 |
|
1p13.2 |
NRAS |
Q61K |
12.50% |
G |
T |
10.7090465 |
12.1263395 |
|
3q26.3 |
PI3KCA |
H1047R |
17.50% |
A |
G |
14.9758454 |
15.7051282 |
|
3q26.3 |
PI3KCA |
E545K |
8.80% |
G |
A |
9.798995 |
15.6359393 |
二、 上海某生物公司H*
實(shí)驗(yàn)樣本：人gDNA
樣本數(shù)目：1（不同Input以及不同酶切時(shí)間）
實(shí)驗(yàn)結(jié)論：1）Input DNA 500 ng/700 ng，PCR 5cycles，未文庫產(chǎn)量 3μg左右 （酶切 12 min，文庫主峰 300-600 bp；酶切 15min，文庫主峰在 300-400 bp）
2）0.78×/ 0.2×雙輪分選，文庫產(chǎn)量 1 μg左右 （主峰 300 bp，文庫范圍 250-450 bp），可滿足客戶實(shí)驗(yàn)需求。
|
實(shí)驗(yàn)參數(shù) |
1 |
2 |
3 |
4 |
|
Input DNA |
500 ng |
500 ng |
700 ng |
700 ng |
|
酶切時(shí)間 |
12 min |
15 min |
12 min |
15 min |
|
Cycles |
5 |
5 |
5 |
5 |
|
文庫產(chǎn)量(μg) |
3.06 |
3.06 |
2.98 |
3.36 |
|
3.12 |
2.91 |
3 |
3 |
|
|
分選后產(chǎn)量(μg) |
0.88 |
0.9 |
0.99 |
0.96 |
|
0.97 |
1.1 |
1.00 |
1.11 |
訂購詳情
|
建庫試劑盒 |
產(chǎn)品編號(hào) |
規(guī)格 |
|
Hieff NGS® Ultima Dual-mode mRNA Library Prep Kit for Illumina® |
12301ES24/96/98 |
24/96/1000 T |
|
12199ES24/96 |
24/96 T |
|
|
12201ES24/96 |
24/96 T |
|
|
12203ES24/96 |
24/96 T |
|
|
12204ES24/96 |
24/96 T |
|
|
12205ES24/96 |
24/96 T |
|
|
12206ES24/96 |
24/96 T |
|
|
文庫構(gòu)建磁珠 |
產(chǎn)品編號(hào) |
規(guī)格 |
|
12599ES08/56 |
5/60 mL |
|
|
12600ES08/56 |
5/60 mL |
|
|
Hieff NGS® DNA Selection Beads（Superior AMPure XP alternative） |
12601ES08/56 |
5/60 mL |
|
12602ES08/56 |
5/60 mL |
|
|
12603ES24/96 |
24/96 T |
|
|
建庫接頭 |
產(chǎn)品編號(hào) |
規(guī)格 |
|
Hieff NGS® 96 Single Index Primer Kit for Illumina®, Set 1/2 |
12611/12612ES02 |
48×2 T |
|
12412/12413ES02 |
96×2 T |
|
|
12414/12415ES02 |
96×2 T |
|
|
12615/12616ES04/16 |
12×4/12×16 T |
|
|
12617/12618ES04/16 |
12×4/12×16 T |
|
|
12610ES96 |
96 T |
|
|
建庫模塊 |
產(chǎn)品編號(hào) |
規(guī)格 |
|
12608ES24/96 |
24/96 T |
|
|
12620ES24/96 |
24/96 T |
|
|
2×Super Canace®Ⅱ High-Fidelity Mix for Library Amplification |
12621ES03/08 |
24/96 T |
|
Hieff NGS® Dual-Mode cDNA Synthesis Kit |
12250ES24/96 |
24/96 T |
|
文庫定量 |
產(chǎn)品編號(hào) |
規(guī)格 |
|
Hieff NGS® Library Quantification Kit for Illumina®, qPCR Master Mix |
12302ES05 |
500 T |
|
Hieff NGS® Library Quantification Kit for Illumina®, DNA Standard (1-6) |
12307ES09 |
6×96 μL |
|
12640ES60/76 |
100/500 T |
|
|
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