在约20%-30%的急性髓系白血病（acute myeloid leukemia,AML）患者中FLT3基因中外显子14和15存在内部串联重复（FLT3 in-tema]tandemduplication，FLT3-ITD）

突变插入的长度在15bp-300bp之间

使用pindel进行检测，限定的范围可设定为chr13:28608000-28608600（hg19）,最小支持的变异reads数目为5(unique reads)

提取所有比对这个区域的reads可以使用Phrap进行拼接，并使用blast与限定范围内的参考序列进行比对

如果出现GAP比对并且，GAP的长度大于等于10bp,则认为该区域存在ITD insertion

pindel在参考文献[1]中提到该软件可以发现频率低到1%的变异,检测性能优越

pindel给出的结果分类如下::

    D = deletionSI = short insertionINV = inversionTD = tandem duplicationLI = large insertionBP = unassigned breakpointsThe reported larger insertion (LI) record is rather different than other types of variants.

pindel给出的结果说明(参考链接：http://gmt.genome.wustl.edu/packages/pindel/user-manual.html) ::

    indextype(LI)ChrIDchrNameleft breakpointnumber of supporting reads for the left coordinateright breakpointnumber of supporting reads for the right coordinate

生信检测示意图如下

PCR验证的引物序列如下[2] ::

    ITD11F             GCAATTTAGGTATGAAAGCCAGC12R             CTTTCAGCATTTTGACGGCAACC12R-FAM         CTTTCAGCATTTTGACGGCAACC11F-HEX	        GCAATTTAGGTATGAAAGCCAGC

另外推荐另一款生信检测软件[3]
getITD https://github.com/tjblaette/getitd

参考文献

1:Detection of FLT3 Internal Tandem Duplication in Targeted, Short-Read-Length, Next-Generation Sequencing Data

2:Establishment of a cost-effective method to detect FLT-ITD and D835 mutations in acute myeloid leukemia patients in the Taiwanese population

3:getITD for FLT3-ITD-based MRD monitoring in AML