j*0
2 楼
要检测病人中某个基因有无突变,8kb,100个case。计划用常规测序方法,但我有一个
问题。
如果是突变是杂合子,那PCR扩增的模板是随机的,有可能扩增出来正常的,可有可能
扩增出来突变的那条链,那测序的时候怎么能区分出来呢?
多谢。
问题。
如果是突变是杂合子,那PCR扩增的模板是随机的,有可能扩增出来正常的,可有可能
扩增出来突变的那条链,那测序的时候怎么能区分出来呢?
多谢。
a*8
3 楼
刚才还有报绿的
f*u
5 楼
要看pd
s*e
7 楼
PD 是7/26/2007
l*o
8 楼
问题是当两个ratio差别大的时候, 肉眼也看不出来.
可以克隆到载体上, 随机挑10个, 分别测序.我以前就这么做过.
可以克隆到载体上, 随机挑10个, 分别测序.我以前就这么做过.
g*g
9 楼
理论上下个月之前都有可能批。
l*1
14 楼
Bingo
plus NGS SNP Bioinformatics platform:
i.e:
http://www.snps3d.org/
more try,
HTTP: //www.snps3d.org/modules.php?name=Go
HTTP: //www.snps3d.org/modules.php?name=Browser
HTTP: //www.snps3d.org/modules.php?name=SNPtargets
HTTP: //www.snps3d.org/modules.php?name=BrowserAll&id=A
from
Valencia A et al. (2012)
Getting personalized cancer genome analysis into
the clinic: the challenges in bioinformatics
Genome Med.4: 61.
web link.
HTTP ://www.ncbi.nlm.nih.gov/pubmed/22839973
【在 G***y 的大作中提到】
: You can directly sequnence the PCR product (mixture of both).
plus NGS SNP Bioinformatics platform:
i.e:
http://www.snps3d.org/
more try,
HTTP: //www.snps3d.org/modules.php?name=Go
HTTP: //www.snps3d.org/modules.php?name=Browser
HTTP: //www.snps3d.org/modules.php?name=SNPtargets
HTTP: //www.snps3d.org/modules.php?name=BrowserAll&id=A
from
Valencia A et al. (2012)
Getting personalized cancer genome analysis into
the clinic: the challenges in bioinformatics
Genome Med.4: 61.
web link.
HTTP ://www.ncbi.nlm.nih.gov/pubmed/22839973
【在 G***y 的大作中提到】
: You can directly sequnence the PCR product (mixture of both).
f*2
15 楼
Recommend using PacBio sequencer. multiplex all 100 cases with barcodes, PCR
amplify the whole 8Kb. A single smrt cell can generate enough sequence data
for the mutation detection. better than regular Sanger sequencing.
Contact me if you need more info.
【在 j**********0 的大作中提到】
: 要检测病人中某个基因有无突变,8kb,100个case。计划用常规测序方法,但我有一个
: 问题。
: 如果是突变是杂合子,那PCR扩增的模板是随机的,有可能扩增出来正常的,可有可能
: 扩增出来突变的那条链,那测序的时候怎么能区分出来呢?
: 多谢。
amplify the whole 8Kb. A single smrt cell can generate enough sequence data
for the mutation detection. better than regular Sanger sequencing.
Contact me if you need more info.
【在 j**********0 的大作中提到】
: 要检测病人中某个基因有无突变,8kb,100个case。计划用常规测序方法,但我有一个
: 问题。
: 如果是突变是杂合子,那PCR扩增的模板是随机的,有可能扩增出来正常的,可有可能
: 扩增出来突变的那条链,那测序的时候怎么能区分出来呢?
: 多谢。
s*s
16 楼
要求科普。pacbio的multiplex这么容易做么?
PCR
data
【在 f*******2 的大作中提到】
: Recommend using PacBio sequencer. multiplex all 100 cases with barcodes, PCR
: amplify the whole 8Kb. A single smrt cell can generate enough sequence data
: for the mutation detection. better than regular Sanger sequencing.
: Contact me if you need more info.
PCR
data
【在 f*******2 的大作中提到】
: Recommend using PacBio sequencer. multiplex all 100 cases with barcodes, PCR
: amplify the whole 8Kb. A single smrt cell can generate enough sequence data
: for the mutation detection. better than regular Sanger sequencing.
: Contact me if you need more info.
f*2
18 楼
对于LZ的项目,用PacBio的成本要比Sanger sequencing少。
multiplex是在PCR step. Barcodes can be added at the 5' end of both forward
and reverse primers for each sample. After PCR, purify and pool the
amplicons together with equal amount. Making one PacBio library with the
pooled sample and run one SMRT cell. Total price about 500 for PacBio
sequencing. The total throughput is about 100Mb per smrt cell with about 20-
50K reads.
LZ的200个PCR pool, 每个至少有100x coverage. 应该可以检测到50% or 100%突变.
【在 j****x 的大作中提到】
: 同求科普,关键是成本
multiplex是在PCR step. Barcodes can be added at the 5' end of both forward
and reverse primers for each sample. After PCR, purify and pool the
amplicons together with equal amount. Making one PacBio library with the
pooled sample and run one SMRT cell. Total price about 500 for PacBio
sequencing. The total throughput is about 100Mb per smrt cell with about 20-
50K reads.
LZ的200个PCR pool, 每个至少有100x coverage. 应该可以检测到50% or 100%突变.
【在 j****x 的大作中提到】
: 同求科普,关键是成本
s*s
19 楼
我还以为你这个比其他ngs的multiplex简单多了呢。看上去还是差不多。
你这个要订200对完全不同的PCR引物做PCR并且纯化,不太靠谱。
我前面说过了,除非一直有这样的sample做,否则multiplex就是自己找麻烦
20-
【在 f*******2 的大作中提到】
: 对于LZ的项目,用PacBio的成本要比Sanger sequencing少。
: multiplex是在PCR step. Barcodes can be added at the 5' end of both forward
: and reverse primers for each sample. After PCR, purify and pool the
: amplicons together with equal amount. Making one PacBio library with the
: pooled sample and run one SMRT cell. Total price about 500 for PacBio
: sequencing. The total throughput is about 100Mb per smrt cell with about 20-
: 50K reads.
: LZ的200个PCR pool, 每个至少有100x coverage. 应该可以检测到50% or 100%突变.
你这个要订200对完全不同的PCR引物做PCR并且纯化,不太靠谱。
我前面说过了,除非一直有这样的sample做,否则multiplex就是自己找麻烦
20-
【在 f*******2 的大作中提到】
: 对于LZ的项目,用PacBio的成本要比Sanger sequencing少。
: multiplex是在PCR step. Barcodes can be added at the 5' end of both forward
: and reverse primers for each sample. After PCR, purify and pool the
: amplicons together with equal amount. Making one PacBio library with the
: pooled sample and run one SMRT cell. Total price about 500 for PacBio
: sequencing. The total throughput is about 100Mb per smrt cell with about 20-
: 50K reads.
: LZ的200个PCR pool, 每个至少有100x coverage. 应该可以检测到50% or 100%突变.
s*s
22 楼
f*2
24 楼
主要有三个方面的改进:
1。硬件上光学系统,升级后可以同时检测15万 ZMWs,通量增加俩倍:200-500Mb per
smrt cell
2。更好的DNA Polymerase,可以跑最长两个小时,增加了average read length up to
5Kb.
3。分析软件的更新,可以只用PAcBio data for de novo assembly, targeted
resequencing for variants detection and DNA modification detection.
【在 N****n 的大作中提到】
: PacBio Sales Start to Pick Up as Company Delivers on Product Enhancements
: What enhancements did they put up? Any comments?
1。硬件上光学系统,升级后可以同时检测15万 ZMWs,通量增加俩倍:200-500Mb per
smrt cell
2。更好的DNA Polymerase,可以跑最长两个小时,增加了average read length up to
5Kb.
3。分析软件的更新,可以只用PAcBio data for de novo assembly, targeted
resequencing for variants detection and DNA modification detection.
【在 N****n 的大作中提到】
: PacBio Sales Start to Pick Up as Company Delivers on Product Enhancements
: What enhancements did they put up? Any comments?
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