Pre-processing methods

IMPI requires as input paired-end reads in *.fastq file format. IMPI allows for processing single files and batches of *.fastq files. For pre-processing IMPI requires a reference gene sequence (nt) and forward and reverse primers. Primers can contain unique molecular identifiers (UMIs).

Data pre-processing can be invoked by clicking on the Start... button

Data input sections for batches of files (left - enter path to folder containing target files) or single file input (right) in paired-end *.fastq file format. Additionally, the reference gene (nt-sequence or in *.fasta file format) and the primer sequences are mandatory input for successful pre-processing. UMI nt sequence should be attached to primer sequence using N.

Read assignments

Overview of the progress of the read counts in IMPI after invoking data pre-processing and application of clusterings.

Read assignment within pre-processing and clustering steps - raw read counts (left), read counts after primer und UMI check (yellow), read counts after merge (green), read counts after excluding reads by specific conditions set using the parameter settings (blue), read counts after UMI assembling (violet) and UMI assembling II (gray)

PWM information

Specific features are extracted and calculated from the SAM output file derived from Bowtie2 mapping and stored in a tab-delimited .csv file. The following features are extracted and used for calculating the position weight matrices later-on in the Allele frequency analysis.

Feature Name	Description
ID	Every read in a FASTQ file has a sequence identifier - this line commonly begins with an ‘@’ character followed by the sequence identifier
UMI	Extracted unique molecular identifier sequence which was part of the primer
UMI_Phred_Quality	Phred quality scores of the UMI encoded in ASCII characters
UMI_Avg_Score	Average Phred quality score of the UMI
Seq	Nucleotide sequence of the read with its deletions and insertions which are defined by the Concise Idiosyncratic Gapped Alignment Report (CIGAR) information
Phred_Quality	Phred quality scores of the whole nucleotide sequence encoded in ASCII characters
Avg_Score	Average Phred quality score of the sequence
Start	Start position of the sequence within the reference gene sequence
Length	Length of the sequence
Insertions	Number of insertions
Deletions	Number of deletions
RefIdentical	Identity of the sequence with the reference gene sequence
RefMismatches	Number of mismatches in comparison with the reference gene sequence
aaSeq	Translated nucleotide sequence to get the amino acid sequence
aaRefIdentical	Identity of the amino acid sequence with the amino acid reference sequence
aaRefMismatches	Number of mismatches in comparison with the amino acid reference sequence