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6.3 years ago
rse
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100
Hi,
I am visualizing alignments using IGV. I want to ask what do you mean by Insert? Is insert size a large unsequenced segment sitting right in between the two reads which escapes sequencing and also shearing during library prep stage? How to validate the existence of a large insert such as 3Mb or 17 Mb as it cannot be confirmed even by a high fidelity Taq (max limit of Genomic DNA pcr is 1-2kb for taq and upto 30kb for hf Taq)?
Regards
Insert sizes are best described by image in this post ( A: What is the different between Read and Fragment in RNA-seq? ).
This question is borderline connected to bioinformatics. You may want to post this over at SeqAnswers.com
Thank you for your reply, but i am still unclear whether the insert refers to the unsequenced DNA segment b/w the 2 reads?
Insert in NGS libraries refers to the entire fragment being sequenced. One samples it from both ends. One can have large inserts (in mate-pair libraries, I am not sure of the upper limit but it would not be Mb more like kb) in special cases.
Insert in your case is an inserted (extraneous?) fragment. You won't be able to see it in your sequence data unless you knew the sequence of that insert before-hand and had reads that tile across the entire region. You could try to do an assembly of your data and see if you are able to see the large insertion by aligning the assembly (using blat/lastz etc) to the available reference.
x-posted: http://seqanswers.com/forums/showthread.php?t=79825
Also previously posted here: IGV Visualization Insert Size