AmpUMI: design and analysis of unique molecular identifiers for deep amplicon sequencing

Abstract

MotivationUnique molecular identifiers (UMIs) are added to DNA fragments before PCR amplification to discriminate between alleles arising from the same genomic locus and sequencing reads produced by PCR amplification. While computational methods have been developed to take into account UMI information in genome-wide and single-cell sequencing studies, they are not designed for modern amplicon-based sequencing experiments, especially in cases of high allelic diversity. Importantly, no guidelines are provided for the design of optimal UMI length for amplicon-based sequencing experiments.ResultsBased on the total number of DNA fragments and the distribution of allele frequencies, we present a model for the determination of the minimum UMI length required to prevent UMI collisions and reduce allelic distortion. We also introduce a user-friendly software tool called AmpUMI to assist in the design and the analysis of UMI-based amplicon sequencing studies. AmpUMI provides quality control metrics on frequency and quality of UMIs, and trims and deduplicates amplicon sequences with user specified parameters for use in downstream analysis.Availability and implementationAmpUMI is open-source and freely available at http://github.com/pinellolab/AmpUMI.

Short DNA Probes Developed for Sample Tracking and Quality Assurance in Gene Panel Testing

“…Although approximately 20% lowered ratios were observed ( Figure 4G), even approximately 0.1% contamination could be reproducibly detected, and these detected values appeared to have good linearity (r Z 0.999). These results suggest that, without the use of a unified molecular identifier, 34 it would be technically difficult to remove PCR bias from quantitative evaluation in this amplicon sequencing case. Nevertheless, these data may support the usefulness of this method for tracking the source of a sample and evaluating cross contamination via an experimental workflow for GPS.…”

Quantifying Hematopoietic Stem Cell Clonal Diversity by Selecting Informative Amplicon Barcodes

“…UMI-based PCR techniques reduce sequencing error by generating consensus alleles from reads with identical UMIs and mitigate PCR bias by reducing read groups with the same UMI to a single deduplicated read 27 . This protocol incorporates a series of annealing/extension steps to tag single genomic DNA template molecules with the UMI barcode.…”

“…The published bioinformatic analyses do not provide a means to identify these uninformative variants or a systematic approach to exclude samples with a low fraction of informative barcodes. Unique molecular identifiers (UMIs) have been used for sequencing error, PCR error, and PCR bias correction 27 , however the UMI PCR protocol more than doubles the sample preparation time and reagent cost, and the extent to which UMIs improve accuracy over standard PCR has not been demonstrated in this setting.…”

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“…There are three reputable databases [1,39,46] that compile accurate information on BRCA1/2 variants classification based on multiple evidences from different sources combined into a Bayesian model to generate a posterior probability and validated by an expert panel [28]: ClinVar, BRCA Exchange, and BRCA Share. In ClinVar, the classification is reliable only if the "review status" contains three stars, meaning that it has been validated by the ENIGMA [25] expert panel, while BRCA Exchange [1] provides the same classification as ClinVar in a more userfriendly environment and more detailed information [14], including a mobile app that provides reclassification updates on variants of interest. On the other hand, BRCA Share™ [7] compiles data from 16 academic laboratories performing BRCA1/2 testing in France.…”