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Library Troubleshooting

Performance Issues
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Things which can cause libraries to perform badly (or not at all) on a Deep Sequencing Run:

Nicked DNA:

The clusters, beads, foci, or whatever they are called depending on the platform in use, are built with single stranded DNA. When you submit a library, though it's usually double stranded and built by PCR amplification, the strands are separated and single strands of DNA are used to build the clusters/template. If the DNA is nicked from the shearing, gel isolation and ETBR exposure, degradation, etc. then the required sequences at each end will not be present and a completed cluster will not be formed. To avoid nicking we recommend using SYBR to stain gels when doing gel purification and sizing. We are also impressed with the new product from NEB called PreCR which repairs nicks very reliably.

Wide size range or several concentrated sizes in a mixture:

This results in a range of cluster sizes. A wide smear of sizes gives clusters of variable sizes and densities which is hard to image accurately. If you have a situation where you cannot size cut your library and request that we run at a lower density to compensate, we will be glad to, however note that your total sequence yield may be greatly reduced. If you have a multiphasic library, e.g. sizes centered around 2 or more different ranges, you will have cluster populations in 2 or more different sizes of consistent densities. The farther apart they are in size the more likely the detection software is to have problems. There are work arounds for this which generally involve making sub-libraries, please talk with us or one of the vendor-provided tech support folks.

Modified DNA:

Abasic sites created by some Taq Polymerases will prevent amplification. Modifications which interfere with annealing, amplification or change the Tm can also prevent good cluster formation. The LACK of the modifications on the Illumina primers (which have proprietary LNA modifications) will result in fewer clusters, so if you are making your own please note this and pay careful attention to the Tm.

Primer Dimer or Primer excess:

These will stick to the positions on the flow cell and give you lots of sequences you really don't want. Gel purification of the final product is highly recommended as is the Topo Cloning to ensure that what goes on the flow cell is indeed what you want. The UMass MBCL provides a coupon for 20 free standard sequencing reactions for each library sequenced in order to encourage and compensate users for doing this important step.

Inhibitor(s) in the sample:

This is most often leftover DNA purification beads or resin transferred into the sample tube and sent along with the finished product. PEG from unwashed Ampure beads can also inhibit cluster formation. TE buffer and other buffers with high levels of EDTA can interfere, please submit in water or EB as requested.

Tips for Improving Libraries
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Extra washes:

An extra bead cleanup after the library is complete can help rid the library of any leftover primer-dimer, help get rid of any inhibitors, and improve cluster seeding in terms of yield and unique reads.

Base Balance:

If you have homogenous sequences in your library insert, you may need phiX to balance the bases. Ideally there should be roughly equal amounts of all 4 bases. At a minimum there must be signal for each laser/light source - A and C are read with one laser, G and T with the other. On instruments like the MiSeq where there is no control lane, lack of base balance can cause the run to terminate prematurely. The same consideration applies when choosing indexes. Picking indexes that are base-balanced will improve the run and help avoid "index-switching" (miss-assignments that occur when indices aren't different enough).

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