The bcl2fastq conversion software can be used to generate FASTQ files from data generated on all current Illumina sequencing systems.įor information on the different settings that can be applied during FASTQ file generation, see the software user guides below. On BaseSpace Sequence Hub, you can find your FASTQ files in the project(s) associated with your run. When analysis completes, the FASTQ files are located in \Data\Intensities\BaseCalls on the MiSeq and \Alignment_#\\Fastq on the MiniSeq.įor all runs uploaded to BaseSpace Sequence Hub, FASTQ file generation automatically occurs after the run is completely uploaded, and the FASTQ files are used as input for the various analysis apps on BaseSpace Sequence Hub. If you need to view a FASTQ file for troubleshooting purposes or out of curiosity, you will need either a text editor that can handle very large files, or access to a Unix or Linux system where large files can be viewed via the command line.įASTQ file generation is the first step for all analysis workflows used by MiSeq Reporter on the MiSeq and Local Run Manager on the MiniSeq. Generally, it is not necessary to view FASTQ files, because they are intermediate output files used as input for tools that perform downstream analysis, such as alignment to a reference or de novo assembly. More detailed information on the FASTQ sequence file format can be found here.įASTQ files can contain up to millions of entries and can be several megabytes or gigabytes in size, which often makes them too large to open in a normal text editor. Here is an example of a single entry in a R1 FASTQ file: These are Phred 33 encoded, using ASCII characters to represent the numerical quality scores. A separator, which is simply a plus ( ) sign.The sequence (the base calls A, C, T, G and N).The exact contents of this line vary by based on the BCL to FASTQ conversion software used. A sequence identifier with information about the sequencing run and the cluster.Each entry in a FASTQ files consists of 4 lines: FASTQ files are compressed and created with the extension *.fastq.gz.įor each cluster that passes filter, a single sequence is written to the corresponding sample’s R1 FASTQ file, and, for a paired-end run, a single sequence is also written to the sample’s R2 FASTQ file. For a paired-end run, one R1 and one Read 2 (R2) FASTQ file is created for each sample for each lane. If samples were not multiplexed, the demultiplexing step does not occur, and, for each flow cell lane, all clusters are assigned to a single sample.įor a single-read run, one Read 1 (R1) FASTQ file is created for each sample per flow cell lane. After demultiplexing, the assembled sequences are written to FASTQ files per sample. Demultiplexing assigns clusters to a sample, based on the cluster’s index sequence(s). If samples were multiplexed, the first step in FASTQ file generation is demultiplexing. This process is called BCL to FASTQ conversion.Ī FASTQ file is a text file that contains the sequence data from the clusters that pass filter on a flow cell (for more information on clusters passing filter, see the “additional information” section of this bulletin). When sequencing completes, the base calls in the BCL files must be converted into sequence data. RTA stores the base call data in the form of individual base call (or BCL) files. During SBS chemistry, for each cluster, base calls are made and stored for every cycle of sequencing by the Real-Time Analysis (RTA) software on the instrument. Illumina sequencing technology uses cluster generation and sequencing by synthesis (SBS) chemistry to sequence millions or billions of clusters on a flow cell, depending on the sequencing platform. The feature set of GCK version 2.5 on both platforms is the same, and Gene Construction Kit files created on either platform can be read by GCK 2.5 on the opposite platform, simplifying collaborations and data. The new version release of Gene Construction Kit is compatible with both Windows and Macintosh platforms.Version 2.5 has been carbonized to run natively in Mac OS X allowing users to take full advantage of the new features of this robust operating system, including the powerful graphics engine, Aqua user interface, and improved stability and performance.A new customizable interface converts all GenBank and EMBL features �one-to-one� to graphical GCK features, enabling molecular biologists to easily work with the latest DNA sequence information available via the Internet.A new Deluxe Importer Module that contains comprehensive features to allow integrated GenBank and EMBL searching and file retrieval over the Internet with importing directly into Gene Construction Kit.- file hosted by Allows graphic manipulation of DNA sequences and sophisticated plasmid drawing options.
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