What are the most common reasons for a sequencing reaction to fail?

Why Does Sequencing Fail?

In the Sequencing Core, we examine virtually every lane from every sample we sequence (excepting only some high-volume bulk jobs where we negotiated minimal technician involvement). We diagnose the problems whenever we can, and some problems just keep coming up again and again.In addition, the Director spends considerable time with clients who have had problems, and carefully examines their experimental design and their protocols for the cause of the failure. The following table is a compilation of the most common failure modes seen in the Advanced Genomics Core.

Here are the most common reasons that sequencing samples fail:

This is SUCH a common problem, I’ll break it down into sub-categories:

  • Inadequate template concentration
    • trying to spec a miniprep plasmid or a PCR product
    • spec readings too low to be accurate
    • inexperience at gel estimation of band intensity
    • failure to check PCR product concentration
    • spec reading with excessive RNA present
    • spec reading with excessive genomic DNA present
  • Overloaded miniprep purification devices (“Loss of Resolution” samples)
  • Failure to streak clones to single-colony
  • Calculation error in primer concentration
  • Multiple priming sites present
  • Multiple inserts present (gives multiple priming or stem-loop)
  • Sequencing incorrect PCR products
  • Failure to fully characterize a new plasmid construct
  • Secondary structure in clone (siRNA constructs, etc)
  • Homopolymer tract (e.g. 3′ sequencing on a cDNA clone)
  • Salt contamination in sample (“ski-slope” effect; typically gel-eluted frags)
  • Mis-paired primers (cross-species primer design, inaccurate sequence basis)

Less common, but still significant:

  • Free dNTPs present in template
  • Primer dimers
  • Primer Tm too low
  • Mixed-up samples and/or primers (i.e. mixed up in the client’s hands).