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  • 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.

  • #2
    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

    Comment


    • #3
      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

      Comment


      • #4
        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.

        Comment


        • #5
          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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          • #6
            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!

            Comment


            • #7
              [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.

              Comment


              • #8
                [QUOTE=AJERYC;88920]
                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, 11:31 PM.

                Comment


                • #9
                  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

                  Comment

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