PanHunter Preprocessing

• 1: Reference genomes

1 - Reference genomes

For the following species reference genomes are available in PanHunter: TO-DO give list

For model species such as mouse and human we maintain several reference genomes. If you would like to know which ensemble version is the latest in the respective release please see our changelog. The reference genome consists of a fasta file that represents the actual DNA strands and an annotation file (gtf file) that contains the positional information of every gene in the genome/fasta file.

For the human reference genome the soft masked (sm) primary assembly is used and this is then extended at Evotec with custom spike-in sequences. Soft-masked means that the nucleotides for repeat stretches are converted to lower-case. Repeats can also be masked (rm), then repeat associated nucleotides are converted to N's. The primary assembly tracks a single unbranched path through the genome. In other words there is only a single base per position and the so called haplotype (alternative base calls) are not included. Toplevel fasta files also include alternative base calls/ haplotype information.

One of the spike-ins, that the soft-masked primary assembly is extended with at Evotec, is PhiX174. PhiX174 is often used in illumina sequencing runs to increase the library quality or balance the GC content (See Phix Illumina version3 - product by illumina). Please note that the reads for PhiX should not be assigned to the fastQ files because there is no index read attached to the PhiX transcripts. Sometimes these reads do get erroneously assigned to a fastQ file due to index read bleeding (the index of a closeby cluster on the flowcell is interpreted as the index of PhiX). Barcode hopping could the other reason for an erronous assignment, this happens when indices break of and reattach within the multiplexed libraries.

The fasta file and gtf annotation contain spike in information for PhiX and for EGFP as well as for 92 ERCC (External RNA Controls Consortium) spike ins that are used to control for variation in RNA sequencing experiments.