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11-Aug-2018 New paper describes octave plots for visualizing alpha diversity.

12-Jun-2018 New paper shows that one in five taxonomy annotations in SILVA and Greengenes are wrong.

18-Apr-2018 New paper shows that taxonomy prediction accuracy is <50% for V4 sequences.

05-Oct-2017 PeerJ paper shows low accuracy of closed- and open-ref. QIIME OTUs.

22-Sep-2017 New paper shows 97% threshold is wrong, OTUs should be 99% full-length 16S, 100% for V4.

24-Nov-2016
UPARSE tutorial video posted on YouTube. Make OTUs from MiSeq reads.

 

USEARCH v11

Chimeras

See also
  Quality control for OTU sequences
  Checking for chimeras in OTU sequences
  Fake chimeras
  Low-divergence chimeras are common

Chimeras are sequences formed from two or more biological sequences joined together. Amplicons with chimeric sequences can form during PCR. Chimeras are rare with shotgun sequencing, but are common in amplicon sequencing when closely related sequences are amplified. Although chimeras can be formed by a number of mechanisms, the majority of chimeras are believed to arise from incomplete extension. During subsequent cycles of PCR, a partially extended strand can bind to a template derived from a different but similar sequence. This then acts as a primer that is extended to form a chimeric sequence ([Smith et al. 2010], [Thompson et al., 2002], [Meyerhans et al., 1990], [Judo et al., 1998][Odelberg, 1995]).

Image

A chimeric template is created during one round, then amplified by subsequent rounds to produce chimeric amplicons. In 16S sequencing, we typically find that only a small fraction of reads is chimeric, perhaps of the order of 1% to 5%. However, when reads are clustered into groups of unique sequences or into OTUs, then we often find that a much larger fraction is chimeric (see Tolstoy's paradox). This is a challenging problem in sequence analysis because chimeras often have low divergence, i.e. are very similar to one of their parents, so are difficult to distinguish from true biological sequences.

It turns out that it is impossible in principle to distinguish chimeras from correct sequences, even when there are no sequence errors and the reference database is complete. This is a very surprising, almost shocking, result which is reported in the UCHIME2 paper. The reason is "fake models", where a correct sequence can be constructed as a chimera from two other correct sequences. Chimeras can have identical sequences to valid genes, so it is impossible for an algorithm to distinguish the two cases from a sequence alone. Fake models are common in practice, hence the problem.
 


Reference (please cite)
R.C. Edgar (2016), UCHIME2: improved chimera prediction for amplicon sequencing, https://doi.org/10.1101/074252
  • UCHIME2 algorithm, improved chimera detection

  • "Fake" chimeras are common, valid biological sequences matching two-parent model

  • Perfect chimera filtering impossible even with complete and correct reference

  • Realistic chimera benchmark