Not all sequence tags are created equal: designing and validating sequence identification tags robust to indels.
Ligating adapters with unique synthetic oligonucleotide sequences (sequence tags) onto individual DNA samples before massively parallel sequencing is a popular and efficient way to obtain sequence data from many individual samples. Tag sequences should be numerous and sufficiently different to ensure sequencing, replication, and oligonucleotide synthesis errors do not cause tags to be unrecoverable or confused. However, many design approaches only protect against substitution errors during sequencing and extant tag sets contain too few tag sequences. We developed an open-source software package to validate sequence tags for conformance to two distance metrics and design sequence tags robust to indel and substitution errors. We use this software package to evaluate several commercial and non-commercial sequence tag sets, design several large sets (max(count) = 7,198) of edit metric sequence tags having different lengths and degrees of error correction, and integrate a subset of these edit metric tags to polymerase chain reaction (PCR) primers and sequencing adapters. We validate a subset of these edit metric tagged PCR primers and sequencing adapters by sequencing on several platforms and subsequent comparison to commercially available alternatives. We find that several commonly used sets of sequence tags or design methodologies used to produce sequence tags do not meet the minimum expectations of their underlying distance metric, and we find that PCR primers and sequencing adapters incorporating edit metric sequence tags designed by our software package perform as well as their commercial counterparts. We suggest that researchers evaluate sequence tags prior to use or evaluate tags that they have been using. The sequence tag sets we design improve on extant sets because they are large, valid across the set, and robust to the suite of substitution, insertion, and deletion errors affecting massively parallel sequencing workflows on all currently used platforms.
Department of Ecology and Evolutionary Biology, University of California Los Angeles, Los Angeles, California, United States of America. brant@faircloth-lab.org
Brant C
BC
Faircloth
Travis C
TC
Glenn
D019295:Computational Biology / Q000379:methods; D004252:DNA Mutational Analysis / Q000379:methods; D020224:Expressed Sequence Tags; D059014:High-Throughput Nucleotide Sequencing; D054643:INDEL Mutation; D020407:Internet; D015203:Reproducibility of Results; D012984:Software