Sequence of Indices (Barcodes) Used for Multiplexing
QuantSeq libraries are intended for a high degree of multiplexing. With QuantSeq for Illumina up to 9,216 samples can be uniquely barcoded in one lane by using the up to 96 external i7 indices (7001-7096) included in the kit together with the 96 external i5 indices (5001-5096), which are part of the Lexogen i5 6 nt Dual Indexing Add-on Kit (Cat. No. 047).
Automated Data Analysis Pipeline
The QuantSeq data analysis pipeline has been implemented on the Bluebee® Genomics Platform and in the Partek Flow software, offering to every user, even without bioinformatics experience, the opportunity to analyze QuantSeq samples in a convenient and fast way.
The pipeline on the Bluebee® Genomics Platform is accessible through a complimentary code provided with each QuantSeq kit. Learn more and get started at https://www.bluebee.com/lexogen/.
Activation codes for additional runs can be purchased from Lexogen via the webshop. Reference genomes for new species may be added upon request. However, this will incur an additional fee. Please enquire about the feasibility of upload for your genome of interest, and pricing via firstname.lastname@example.org.
The QuantSeq pipeline in the Partek Flow software is available for any Partek Flow user at http://www.partek.com/lexogen-quantseq-pipeline.
QuantSeq – FWD Data Analysis
The following analysis pipeline demonstrates a principal workflow.
The used tools are freely available and to a great extend part of the Galaxy platform1. All proprietary Software, for instance CLC workbench, should perform similarly.
The script examples uses mainly the bash internal for-loop; in Galaxy for instance, the single files have to be processed manually one by one.
Following software packages (and their dependencies) should be installed when following the given protocol:
- STAR aligner
Create a working directory and the subdirs fastq, qualitycheck and download the de-multiplexed fastq files into the fastq dir.
Let the fastq files be:
Let your STAR index files be in /data/star/human, the file truseq_rna.fa.gz is provided by bbmap and is here symbolically linked to /data/resources/truseq_rna.fa.gz.
The file /data/resources/polyA.fa.gz is a simple GNU-zipped fasta file containing a single entry with 18 ‘A’s.
1Goecks, J, Nekrutenko, A, Taylor, J and The Galaxy Team. Galaxy: a comprehensive approach for supporting accessible, reproducible, and transparent computational research in the life sciences. Genome Biol. 2010 Aug 25;11(8):R86.