m******p
发帖数: 67 | 1 I have genomic DNA samples of about 1000 diabetic patients. We are
interested in examining the sequence variations of a gene in these samples.
But we are not good at this and have 2 questions.
1) Can we do that by traditional Sanger sequencing? This is very doable.
We first PCR the fragment of interest, and then do sequencing. But the
problem is that Sanger sequencing cannot distinguish paternal and maternal
alleles, that is, we cannot tell whether a variation is homozygous or
heterozygous, unless by looking at the sequencing graph manually, but this
is not practical when thousands of samples are involved.
2) What are available sequencing methods to do that?
Thank you very much! |
m*****7
发帖数: 366 | 2 Depends on your hypothesis, if you want to test just SNPs. Just genotype
some SNPs in your gene, while usually it needs controls, but of course you
can compare it with public available data such as 1000 genomes..
If you want to really "sequence" the gene, I know there are some targeted
sequencing toolkit such as Ion Torrent. In such case you can see the rare
mutation of this gene.
I am not sure where are you and what your university/center usually do,
while I know in our school there is a sequencing core and just give them
sample and money they will do all sequencing for you.
Hope that helps...
.
.
【在 m******p 的大作中提到】
: I have genomic DNA samples of about 1000 diabetic patients. We are
: interested in examining the sequence variations of a gene in these samples.
: But we are not good at this and have 2 questions.
: 1) Can we do that by traditional Sanger sequencing? This is very doable.
: We first PCR the fragment of interest, and then do sequencing. But the
: problem is that Sanger sequencing cannot distinguish paternal and maternal
: alleles, that is, we cannot tell whether a variation is homozygous or
: heterozygous, unless by looking at the sequencing graph manually, but this
: is not practical when thousands of samples are involved.
: 2) What are available sequencing methods to do that?
|
l*****a
发帖数: 1431 | 3 1000个sample，做next generation sequencing吧， 应该比sanger 便宜。 |
c********e
发帖数: 598 | 4
.
.
NGS is expensive.You may try a batch of samples first.
Then you can run probe-based QPCR based on NGS results.
【在 m******p 的大作中提到】
: I have genomic DNA samples of about 1000 diabetic patients. We are
: interested in examining the sequence variations of a gene in these samples.
: But we are not good at this and have 2 questions.
: 1) Can we do that by traditional Sanger sequencing? This is very doable.
: We first PCR the fragment of interest, and then do sequencing. But the
: problem is that Sanger sequencing cannot distinguish paternal and maternal
: alleles, that is, we cannot tell whether a variation is homozygous or
: heterozygous, unless by looking at the sequencing graph manually, but this
: is not practical when thousands of samples are involved.
: 2) What are available sequencing methods to do that?
|
b****r
发帖数: 17995 | 5 很耐心。基本同意
【在 m*****7 的大作中提到】
: Depends on your hypothesis, if you want to test just SNPs. Just genotype
: some SNPs in your gene, while usually it needs controls, but of course you
: can compare it with public available data such as 1000 genomes..
: If you want to really "sequence" the gene, I know there are some targeted
: sequencing toolkit such as Ion Torrent. In such case you can see the rare
: mutation of this gene.
: I am not sure where are you and what your university/center usually do,
: while I know in our school there is a sequencing core and just give them
: sample and money they will do all sequencing for you.
: Hope that helps...
|
n****c
发帖数: 42 | 6 If you do NGS, one MiSeq run will be more than enough. ~$1-2k for the
sequencing. Cheaper than Sanger for sure. |
m******p
发帖数: 67 | 7 thank you very much for the suggestions.
1) now we have already got lots of sequencing data by traditional Sanger
sequencing. can we still use this data to do dome meaningful analysis and
conclusion?
2) 'targeted sequencing toolkit such as Ion Torrent' appears to be able to
distinguish alleles (het vs. homo). Do you know which companies can do this?
again, thank you very much. |
s*****3
发帖数: 20 | 8 Here are my 2 cents.
1) If you only look at one or a few SNP(s) for that gene, Taqman SNP
analysis (in 384-well or even in 96-well format) can be your choice. This is
much
cheaper and faster than Sanger or NGS.
2) If you look at 10-100 SNPs, Openarray SNP analysis (using OpenArray or
Quantstudio) can be your choice.
3) If you look at a large number of SNPs, e.g >100 SNPS (though I doubt it
since you only look at one gene), you could consider NGS target resequencing
such as Ion Torrent custom Ampliseq.
No matter which of the 3 above platforms you pick, your pilot experiment
results collected using Sanger are important and can be used to validate
the assay using the platform of choice. Your Sanger results can be used to
distinguish homozygous (one peak) from heterozygous (two peaks) variants but
cannot be utilized to distinguish paternal vs maternal alleles. |
m******p
发帖数: 67 | 9 srr8623 - thanks a lot for the insightful suggestions. |
m**********2
发帖数: 2646 | 10 You can use the Seqscape software (by applied biosystem I think). You can
put in all the exon information of this gene and import all the sanger
sequencing files at the same time. It will detect the SNPs right away (you
can set the threshhold, for example,call any mutation that is at 25% of the
WT in peak size).
After they made the SNP calls, you can click on each SNP to look at the peak
and determine whether it is homozygous or heterozygous.
The software cost about $4000~6000, but it has a 30-day free trial. |
s*****3
发帖数: 20 | 11 milandream82--Thank you for sharing information about Seqscape. Do you know
if the software works for somatic mutation analysis of Sanger sequencing?
I was wondering if it could automatically call mutations at 20% or lower
percentage (if so, what is the limit of detection?). Thanks in advance. |
