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Input

To upload files, drag and drop all files into the upload box, or select your files by first clicking the Select Files button.

Tip

Submit all files for your analysis together (ie. multiple samples) to speed up processing time and perform cross-sample analyses.

Format

BugSeq supports all standard sequencing file formats. The following file formats are currently accepted:

  • FAST5: Raw nanopore sequencing data

    • Acceptable file extensions: .fast5
  • FASTQ: Basecalled nanopore or Illumina sequences

    • Acceptable file extensions:
      • Uncompressed: .fq, .fastq
      • Compressed: .fq.gz, .fastq.gz
  • FASTA: Nucleic acid sequences without quality scores

    • Acceptable file extensions:
      • Uncompressed: .fa, .fna and .fasta
      • Compressed: .fa.gz, .fna.gz and .fasta.gz
    • ❗ Note: BugSeq currently only accepts FASTA sequences from PacBio HiFi sequencing experiments.

Tip

BugSeq performs best with .fast5 files from nanopore sequencers and .fastq.gz files from Illumina sequencers.

Experimental Design

Platform

You may submit files from any Oxford Nanopore, PacBio or Illumina sequencer.

BugSeq will automatically detect the sequencing platform and perform tailored, best-practice analyses. The following processes are adjusted based on platform:

  • Basecalling
  • Demultiplexing
  • Quality evaluation
  • Adapter trimming
  • Quality trimming and filtering
  • Metagenomic classification
  • Alignment
  • Assembly

Sequencing Strategy

BugSeq supports all widely accepted sequencing strategies. These include:

  • Amplicon Sequencing

    • 16S/ITS
    • MLST
    • Viral amplicons
      • SARS-CoV-2: ARTICv1, ARTICv2, ARTICv3
      • Custom primer schemes - get in touch.
  • Whole Genome Sequencing (WGS)

  • Metagenomic Sequencing

Sequenced material can be DNA or RNA. Nucleic acid can be sequenced directly or amplified with techniques such as PCR.

Multiplexing

BugSeq automatically performs demultiplexing and adapter trimming on nanopore sequencing data using a custom version of qcat, expanded to support ONT’s latest sequencing kits. Samples are named based on the sequencing run identification and barcode. These fields are contained in nanopore FASTQ headers by default.

Skipping demultiplexing

BugSeq parses FASTQ headers for multiplexing data. Occasionally, users may want to skip this split (eg. if you have multiple run IDs from pausing and restarting the sequencer in the middle of a run). To process samples without splitting them by run ID and barcode, strip this information from read headers before submitting:

head -n 2 WILL_BE_SPLIT.fastq

@71cc9a9f-139a-4f6b-afef-a5c3dacbf9f4 runid=68739378-ec00-4fef-ae41-02067870808e barcode=1
ATGCATGC

cut -f 1 -d ' ' WILL_BE_SPLIT.fastq > NOT_SPLIT.fastq

head -n 2 NOT_SPLIT.fastq
@71cc9a9f-139a-4f6b-afef-a5c3dacbf9f4
ATGCATGC

New Designs

Don’t see your experimental design combination here? Get in touch with us via support.


Last update: May 14, 2021