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Old 09-22-2011, 01:43 PM   #1
arvi8689
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Default Exome sequencing

Hey I am trying to sequence the melanoma exome to find somatic mutation involved in the melanoma. I am very new to this technique and I have some questions regarding this:

1)Generally how many exomes from different melanoma samples need to be done to get some satisfactory results?
2)What coverage is essential for such a study? Initially I was thinking of around 30X but some papers have used nearly 100X. Will I be okay with 30X coverage of the exome?

Your help is well appreciated.
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Old 04-22-2012, 11:15 PM   #2
hagir
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hello every body, i'm a new member and hope that you help me
i want to perform an exome sequencing on oral cancer cases using illumina. i need you to help me with a good protocol for doing so
thanks
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Old 04-22-2012, 11:15 PM   #3
hagir
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Default exome sequencing

hello every body, i'm a new member and hope that you help me
i want to perform an exome sequencing on oral cancer cases using illumina. i need you to help me with a good protocol for doing so
thanks
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Old 05-05-2012, 10:56 AM   #4
AJERYC
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30x is definitely not enough, since it means 30x averge, so you will get a lot of regions with very few sequences, that will make impossible for you to resolve heterozygous mutations. Go 50x or 100x.
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Old 05-15-2012, 06:22 PM   #5
ymc
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Quote:
Originally Posted by AJERYC View Post
30x is definitely not enough, since it means 30x averge, so you will get a lot of regions with very few sequences, that will make impossible for you to resolve heterozygous mutations. Go 50x or 100x.
How many x also depends on your enrichment kit. For example, according to the BGI paper, for 90% call sensitivity, Nimblegen SeqCap EZ v1 needs 50x, Agilent SureSelect All Exon needs 80x.
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Old 11-08-2012, 11:26 PM   #6
gensdei
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Cell lines have the benefit of being a relatively pure population of clonal cells and thus represent a particular snapshot of cancers. Is there anybody who have seen a paper using exome-seq with 100x coverage based on cell line? If there is not, does it means that we don't need perform that deep sequencing due to its purity?

Thanks in advance!
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Old 11-09-2012, 01:20 AM   #7
AJERYC
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[QUOTE=gensdei;88907]Cell lines have the benefit of being a relatively pure population of clonal cells and thus represent a particular snapshot of cancers. Is there anybody who have seen a paper using exome-seq with 100x coverage based on cell line? If there is not, does it means that we don't need perform that deep sequencing due to its purity?


Theoretically a cell line is homozygous and that is more easy to resolve with NGS so you could go 10-30x.
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Old 11-09-2012, 02:44 AM   #8
gensdei
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[QUOTE=AJERYC;88920]
Quote:
Originally Posted by gensdei View Post
Cell lines have the benefit of being a relatively pure population of clonal cells and thus represent a particular snapshot of cancers. Is there anybody who have seen a paper using exome-seq with 100x coverage based on cell line? If there is not, does it means that we don't need perform that deep sequencing due to its purity?


Theoretically a cell line is homozygous and that is more easy to resolve with NGS so you could go 10-30x.
To be more specific, exome-seq from cell line have no variant sites, e.g, SNP? Theoretically is it true? If so, you are right, no need for deep sequencing. What about exome-seq from cancer cell line? Did anybody see a paper on 100x exome-seq data from cancer cell line? Thanks.

Last edited by gensdei; 11-10-2012 at 10:31 PM.
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Old 11-12-2012, 01:24 AM   #9
mamons
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Quote:
Originally Posted by hagir View Post
hello every body, i'm a new member and hope that you help me
i want to perform an exome sequencing on oral cancer cases using illumina. i need you to help me with a good protocol for doing so
thanks
http://seqanswers.com/wiki/How-to/exome_analysis
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