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Usage

Dependencies

  • Julia >= 1.10 (includes the Distributed standard library)
  • Julia packages
    • DataFrames >= 1.7.0
    • CSV >= 0.10.14
    • CodecZlib >= 0.7.0
    • BufferedStreams >= 1.2.2

Basic Usage

The primary function of this package is execute_demultiplexing(). It classifies sequences in a FASTQ file by aligning them with reference barcodes in barcode file. Usage is as follows:

using BioDemuX
execute_demultiplexing(FASTQ_file, barcode_file, output_directory)

Input

FASTQ File

  • There is no restriction on the sequence length in the FASTQ file.
  • The input can be gzipped; the function automatically detects and processes the files accordingly.
  • The function can take one or two FASTQ files as input. In the case of using two FASTQ files, the command can be executed as follows:
execute_demultiplexing(FASTQ_file1, FASTQ_file2, barcode_file, output_directory)

When using two FASTQ files, sequences in the FASTQ_file2 are classified based on the alignment of the FASTQ_file1 sequences with the barcodes in the barcode reference file. Hence, the corresponding reads in both FASTQ files must be in the same order and present in equal numbers.

Barcode Reference File

  • The reference file is expected to be a CSV or TSV file containing the following columns: ID, Full_seq, Full_annotation, as shown below:
ID  Full_seq	Full_annotation
001-barcode ACAGACUACAAA XXXBBBBBBBXX

  • In the Full_seq column, the region specified as B in the Full_annotation column is considered as the barcode.

  • Alternatively, a FASTA file of barcode sequences can be used as the reference. In this case, each sequence in the FASTA file is treated as a full barcode (the entire sequence is considered the barcode region) and the header line of each entry (without the > prefix) is used as its ID.

Output

  • All output files will be saved in the specified output_directory.
  • The output is gzipped depending on the input FASTQ format, or can be specified using the gzip_output option.
  • The names of the output files are based on the filename of the FASTQ file as the prefix and the ID values in the barcode reference file. For example, if the FASTQ filename is sample.fastq and the reference file contains IDs such as 001 and 002, the resulting output files will be named sample.001.fastq, sample.002.fastq, and so on. You can freely change the prefix by specifying the output_prefix argument.
  • Sequences that do not match any barcode in the reference file are saved in unknown.fastq. Sequences that have ambiguous classification (i.e., they match multiple barcodes with similar scores) are saved in ambiguous_classification.fastq. These FASTQ files also have prefix like sample.unknown.fastq and sample.ambiguous_classification.fastq.
  • If the output_directory does not exist, a new directory is created to store the output files.