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Old 12-02-2015, 02:11 PM   #4
Bioo Scientific
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Location: Austin, Tx

Join Date: Oct 2009
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Hi Nucacidhunter,

Thanks for the question. The work by NEB is certainly interesting. It looks like the only original findings are that bias can also be reduced by using either terminal or internal random bases and that efficiency can be improved through co-folding adapters. Other conclusions, like both 3’ and 5’ ligation contributing to bias and the observation that some bias can be explained through secondary structure and co-folding, are mostly just confirmations and more detailed analysis of previous findings. It should also be noted that all of these experiments were performed on a synthetic miRNA pool, so it will be interesting to see how this strategy works with total RNA samples. The strategy implemented for avoiding adapter-dimer formation will be particularly interesting, as adapter-dimer is a much bigger problem with libraries prepared from real-world samples and is more difficult to prevent when using adapters with randomized regions. It will also be interesting to see if other, independent groups confirm these findings, as is the case with the bias reducing effects of using adapters with randomized ends.

One thing that caught our eye was the claim that “Randomized adaptor regions adjacent to the ligation junctions can further complicate sequence analysis if an unknown sample were to be sequenced because the termini of the sRNA in a sequencing read cannot be precisely determined.” As long as the read goes into the adapter, the random bases are easily identified and removed as the first 4 bases of the read and the last 4 bases of the read prior to the adapter sequence (assuming 4 nt random regions were used), allowing precise determination of sRNA ends. And if the insert is too long for the read to go into the adapter sequence, the 3' end cannot be determined no matter whether the random bases were at the ligation junction or internal.

In fact, internal random bases are more likely to complicate sequencing than random bases at the ligation junctions, as the constant region that is read at the beginning of the reads can cause issues related to low diversity. This was illustrated in NEB’s publication when the authors noted that “Two sets of new adaptors (v1 + v2) were used for Illumina sequencing in order to generate sequence diversity for the instrument in the 7 base region between the randomized region and RNA insert...” The only other comparison of randomized ends versus internal randomization in reducing bias concludes that “Surprisingly, the improvement in sequencing results was similar when either the 3’ Rand or 3 ’MidRand adaptor were used.” Thus, it appears that NEB’s internal randomization strategy does not provide any advantages over end randomization.

At Bioo Scientific we have long recognized the importance of reducing bias in small RNA library prep, resulting in last year's launch of the NEXTflex™ Small RNA Sequencing Kit v2, which uses randomized adapters to reduce ligation bias. We followed that up with the recent launch of the NEXTflex™ Small RNA Seq Kit v3, which combines bias reduction with completely gel-free or low-input protocols. These kits are currently available, providing scientists with optimized kits for reduced-bias small RNA library preparation.
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