Exome sequencing

[arvi8689]

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.

[hagir]

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

[hagir]

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

[AJERYC]

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.

[ymc]

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.

[gensdei]

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!

[AJERYC]

[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.

[gensdei]

[QUOTE=AJERYC;88920]

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.

[mamons]

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