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The yield obtained from next generation sequencers has increased almost exponentially in recent years, making sample multiplexing common practice. While barcodes (known sequences of fixed length) primarily encode the sample identity of sequenced DNA fragments, barcodes made of random sequences (Unique Molecular Identifier or UMIs) are often used to distinguish between PCR duplicates and transcript abundance in, for example, single-cell RNA sequencing (scRNA-seq). In paired-end sequencing, different barcodes can be inserted at each fragment end to either increase the number of multiplexed samples in the library or to use one of the barcodes as UMI. Alternatively, UMIs can be combined with the sample barcodes into composite barcodes, or with standard Illumina® indexing. Subsequent analysis must take read duplicates and sample identity into account, by identifying UMIs.
Barcoding Strategies
a Schematic view of the multiplexed library processing. A unique and different barcode (BC, white box with black stripes) is used for each sample. The barcode is placed further down the DNA fragment and sequenced in a specific sequencing round (Illumina® TruSeq™, left); or directly upstream the DNA fragment and sequenced concomitantly (custom protocol, right). After sequencing and image processing, reads of multiplexed samples are mixed together in the fastq result file. For each read, the barcoding sequence (black box with white stripes) is computationally clipped off the read end (custom protocols) or read from the additional barcode file (Illumina® TruSeq™, index file is provided with the I1 option); and the original sample is identified by comparing this barcoding sequence to known barcodes. Finally, read sequences are saved in sample specific fastq files. b In PE sequencing, barcodes can be added to one or both fragment ends. The Je demultiplex BPOS option indicates which read(s) contain(s) the barcode(s). c demultiplex options for barcodes present at both read ends. A decision is needed to specify which barcode is used to identify separate samples. d Combining UMIs (BC1 and BC2, white box with black stripes) with Illumina sample indexing (white box with black dots, top) or as composite barcode (bottom). In a composite barcode, the number of random base upstream and downstream the sample index is variable
Existing tools do not support these complex barcoding configurations and custom code development is frequently required. Here, researcher from the EMBL present Je, a suite of tools that accommodates complex barcoding strategies, extracts UMIs and filters read duplicates taking UMIs into account. Using Je on publicly available scRNA-seq and iCLIP data containing UMIs, the number of unique reads increased by up to 36 %, compared to when UMIs are ignored.
Availability – Je is implemented in JAVA and uses the Picard API. Code, executables and documentation are freely available at http://gbcs.embl.de/Je . Je can also be easily installed in Galaxy through the Galaxy toolshed.
