TETyper

TETyper is a command line tool designed for typing a specific transposable element (TE) of interest from paired-end sequencing data. It determines single nucleotide variants (SNVs) and deletions within the TE, as well as flanking sequences surrounding the TE.

TETyper can be cited as follows:

Sheppard et al bioRxiv 288001; doi: https://doi.org/10.1101/288001

Installation

Requirements:

• python 2.7 or python 3 (with Biopython, pysam, pyvcf)
• samtools, bcftools (tested on version 1.4.1)
• bwa (tested on version 0.7.12)
• spades (tested on version 3.10.1)
• BLAST+ (tested on version 2.2.25)

TETyper is pip-installable, enabling automatic installation of python dependencies:

pip install TETyper

To check whether TETyper is running correctly, a small test dataset has been provided. If the following command is executed:

TETyper.py --ref Tn4401b-1.fasta --fq1 SRR1582895_sm_1.fq.gz --fq2 SRR1582895_sm_2.fq.gz --outprefix test --flank_len 5 --struct_profiles struct_profiles.txt --snp_profiles snp_profiles.txt --show_region 7202-8083

then the file test_summary.txt should look something like this:

Deletions	Structural_variant	SNPs_homozygous	SNPs_heterozygous	Heterozygous_SNP_counts	SNP_variant	Combined_variant	Left_flanks	Right_flanks	Left_flank_counts	Right_flank_counts	7202-8083_presence
none	Tn4401b	C8015T	none	none	2	Tn4401b-2	AGATA|GTTCT	AGATA|GTTCT	56|151	52|101	1

Note that the exact values in the Left_flank_counts and Right_flank_counts columns may vary slightly depending on the bwa version used.

Running TETyper

Basic usage

TETyper.py --ref REFERENCE.fasta --fq1 FORWARD_READS.fq.gz --fq2 REVERSE_READS.fq.gz --outprefix OUTPREFIX --flank_len FLANK_LENGTH

Output

TETyper produces the following output files:

• OUTPREFIX_summary.txt: Summary file containing all typing outputs, described in detail below
• OUTPREFIX.log: Log file containing details of individual steps performed, as well as any errors
• OUTPREFIX.bam: Reads mapped to REFERENCE.bam
• OUTPREFIX_mappedreads_1.fq, OUTPREFIX_mappedreads_2.fq: Mapped reads converted to fastq format
• OUTPREFIX_spades: Directory containing spades assembly from mapped reads
• OUTPREFIX_blast.txt: Tabular blastn results comparing the spades assembly to REFERENCE.fasta
• OUTPREFIX.vcf: SNVs identified

The summary file contains 11-12 columns. These are:

• Deletions: A list of sequence ranges corresponding to regions of the reference classified as deletions for this sample, or "none" for no deletions.
• Structural_variant: If --struct_profiles is specified and the pattern of deletions above corresponds to one of these profiles, then the profile name is given, otherwise "unknown".
• SNPs_homozygous: A list of homozygous SNPs identified, or "none".
• SNPs_heterozygous: A list of heterozygous SNPs identified, or "none".
• Heterozygous_SNP_counts: For each heterozygous SNP, the number of reads supporting the reference and alternative calls, or "none" if there are no heterozygous SNPs.
• SNP_variant: If --snp_profiles is specified and the pattern of homozygous and heterozygous SNPs corresponds to one of these profiles, then the profile name is given. Otherwise "unknown".
• Combined_variant: Single name combining Structural_variant and SNP_variant, separated by "-".
• Left_flanks: A list of distinct sequences passing quality filters that flank the start position of the reference.
• Right_flanks: A list of distinct sequences passing quality filters that flank the end position of the reference.
• Left_flank_counts: The number of high quality reads supporting each of the left flanking sequences.
• Right_flank_counts: The number of high quality reads supporting each of the right flanking sequences.
• X_Y_presence: If --show_region is specified as --show_region X-Y, this column shows 1 if the entirety of that region is classified as present (i.e. no overlap with deleted regions), or 0 otherwise. If --show_region is unspecified, this column is omitted.

Advanced usage (all options):

• -h, --help: show this help message and exit
• --outprefix OUTPREFIX: Prefix to use for output files. Required.
• --ref REF: Reference sequence in fasta format. If not already indexed with bwa, this will be created automatically. A blast database is also required, again this will be created automatically if it does not already exist. Required.
• --refdb REFDB: Blast database corresponding to reference file (this argument is only needed if the blast database was created with a different name).
• --fq1 FQ1: Forward reads. Can be gzipped.
• --fq2 FQ2: Reverse reads. Can be gzipped.
• --fq12 FQ12: Interleaved forward and reverse reads.
• --bam BAM: Bam file containing reads mapped to the given reference. If the reads have already been mapped, this option saves time compared to specifying the reads in fastq format. If this option is specified then --fq* are ignored.
• --assembly ASSEMBLY: Use this assembly (fasta format) for detecting structural variants instead of generating a new one. This option saves time if an assembly is already available.
• --spades_params SPADES_PARAMS: Additional parameters for running spades assembly. Enclose in quotes and precede with a space. Default: " --cov-cutoff auto --disable-rr". Ignored if --assembly is specified.
• --struct_profiles STRUCT_PROFILES: File containing known structural variants. Tab separated format with two columns. First column is variant name. Second column contains a list of sequence ranges representing deletions relative to the reference, or "none" for no deletions. Each range should be written as "startpos-endpos", with multiple ranges ordered by start position and separated by a "|" with no extra whitespace.
• --snp_profiles SNP_PROFILES: File containing known SNP profiles. Tab separated format with three columns. First column: variant name, second column: homozygous SNPs, third column: heterozygous SNPs. SNPs should be written as "refPOSalt", where "POS" is the position of that SNP within the reference, "ref" is the reference base at that position (A, C, G or T), and "alt" is the variant base at that position (A, C, G or T for a homozygous SNP; M, R, W, S, Y or K for a heterozygous SNP). Multiple SNPs of the same type (homozygous or heterozygous) should be ordered by position and separated by a "|". "none" indicates no SNPs of the given type.
• --flank_len FLANK_LEN: Length of flanking region to extract. Required.
• --min_reads MIN_READS: Minimum read number for including a specific flanking sequence. Default 10.
• --min_each_strand MIN_EACH_STRAND: Minimum read number for each strand for including a specific flanking sequence. Default 1.
• --min_mapped_len MIN_MAPPED_LEN: Minimum length of mapping for a read to be used in determining flanking sequences. Higher values are more robust to spurious mapping. Lower values will recover more reads. Default 30.
• --min_qual MIN_QUAL: Minimum quality value across extracted flanking sequence. Default 10.
• --show_region SHOW_REGION: Display presence/absence for a specific region of interest within the reference (e.g. to display blaKPC presence/absence with the Tn4401b-1 reference, use "7202-8083")
• --threads THREADS: Number of threads to use for mapping and assembly steps. Default: 1
• -v, --verbosity: Verbosity level for logging to stderr. 1 = ERROR, 2 = WARNING, 3 = INFO, 4 = DUBUG. Default: 3.
• --no_overwrite: Flag to prevent accidental overwriting of previous output files. In this mode, the pipeline checks for a log file named according to the given output prefix. If it exists then the pipeline exits without modifying any files.

A note on the profile files

The arguments --struct_profiles and --snp_profiles enable the user to specify a naming system for identified variants. This simply provides a convenient way of generating output that is more human-readable than a list of deletions or SNVs. This is probably not helpful when running TETyper for the first time with a new reference, but may be useful once relevant variants have been identified.

Suggested usage for KPC isolates

TETyper was designed with the blaKPC transposon Tn4401 in mind. A Tn4401b reference sequence is provided with TETyper (Tn4401b-1.fasta), as well as example profile definitions for SNVs / deletions with respect to this reference (struct_profiles.txt and snp_profiles.txt). To use these, TETyper can be run as follows:

TETyper.py --ref Tn4401b-1.fasta --fq1 FORWARD_READS.fq.gz --fq2 REVERSE_READS.fq.gz --outprefix OUTPREFIX --flank_len FLANK_LENGTH --struct_profiles struct_profiles.txt --snp_profiles snp_profiles.txt --show_region 7202-8083

Re-running samples

TETyper provides options for specifying the mapped bam file and/or assembly file in order to save processing time if the same samples are rerun with different parameters. For example, newly discovered profiles can be manually appended to the profile files. TETyper can then be rerun with the modified profile files, without redoing all the processing steps, by specifiying the mapped bam file and spades assembly as parameters instead of the original reads. E.g.:

TETyper.py --ref Tn4401b-1.fasta --outprefix RERUN --bam OUTPREFIX.bam --assembly OUTPREFIX_spades/contigs.fasta --flank_len FLANK_LENGTH --struct_profiles STRUCT_PROFILES_MODIFIED.txt --snp_profiles SNP_PROFILES_MODIFIED.txt --show_region 7202-8083

The --bam and --assembly options can also be useful for re-running samples with different parameters for flanking sequence extraction (e.g. a different flank length).