![]() ![]() N characters denote positions which contain randomers. are consistent (positions -6 to -4), but. ![]() The rules governing 3’ barcodes are the same, except that the positions are defined relative to the 3’ end of the read. are not consistent with the first three barcodes (or each other) because, relative to the 5’ end the barcodes are in positions 3-5 and 4-7 respectively. are all consistent because the non-N characters are all in positions 4-6 relative to the 5’ end of the read. Barcodes can have different numbers of Ns, provided that the non-N characters are all consistent. 5’ barcodes do not have to be consistent with 3’ barcodes. 3’ barcodes linked to different 5’ barcodes do not have to be consistent with each other. It is required that the length and position of the non-N nucleotides in the barcodes be consistent for all 5’ barcodes, and all 3’ barcodes linked to that specific 5’ barcode if used. There are certain constraints on the barcode sequences that can be used. For example, the following csv has three 5’ barcodes, the second of which is linked to two 3’ barcodes: Barcode csv (-b, -barcodes):įor the barcode csv, the first column contains a list of all the 5’ barcodes, and (optionally) the other column(s) contain the 3’ barcode(s) linked to that specific 5’ barcode. If your fastq is uncompressed, or compressed in a different format, convert it to gzipped format or Ultraplex will not work. The fastq file should be a 4-line per read fastq. Ultraplex -i your_fastq_ -b your_barcode_csv.csv How do I use Ultraplex? Installation Condaīuild an ultraplex environment straight from our yaml: Furthermore, if you are doing single cell RNA-seq or using, for example, Oxford Nanopore long-read sequencing, there are other softwares specifically designed for these purposes. If instead you are using a commercial library prep kit (eg Illumina Truseq or Lexogen Quantseq) then in all likelihood the sequencing facility will already have demultiplexed the files for you, so Ultraplex is probably not for you. Ultraplex is primarily designed for the demultiplexing of sequencing data generated using in-house library preparation protocols, with custom adaptors (for example iCLIP libraries). It relies heavily on code from the excellent Cutadapt tool. It is capable of demultiplexing an entire HiSeq lane, consisting of ~400 million reads, in just 20 minutes. Ultraplex was designed with speed and ease of use in mind. Writes out files for each barcode (or barcode combination) Moves unique molecular identifiers (UMIs) to the read header for downstream deduplicationĭetects 5’ and (optionally) 3’ barcodes for (combinatorial) demultiplexing Removes sequencing adaptors (eg Illumina universal sequencing adaptor) Removes poor quality bases (normally from the 3' end only) ![]() It performs the following processing steps: Ultraplex is an all-in-one software package for processing and demultiplexing fastq files. ![]()
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