iVar is a computational package that contains functions broadly useful for viral amplicon-based sequencing. Additional tools for metagenomic sequencing are actively being incorporated into iVar. While each of these functions can be accomplished using existing tools, iVar contains an intersection of functionality from multiple tools that are required to call iSNVs and consensus sequences from viral sequencing data across multiple replicates.
Allocate an interactive session and run the program. Sample session:
[user@biowulf]$ sinteractive [user@cn4471 ~]$ module load ivar [+] Loading samtools 1.9 ... [+] Loading ivar 1.3.1 ...Copy sample data to your current folder:
[user@cn4471 ~]$ cp -r $IVAR_DATA/* . [user@cn4471 ~]$ cp test_amplicon.sorted.bam test.bamPreprocess the data with samtools: [user@cn4471 ~]$ samtools sort -o test.sorted.bam test.bam [user@cn4471 ~]$ samtools index test.sorted.bam iVar can be used as follows:
[user@cn4471 ~]$ ivar -h
Usage: ivar [command <trim|callvariants|filtervariants|consensus|getmasked|removereads|version|help>]
Command Description
trim Trim reads in aligned BAM file
variants Call variants from aligned BAM file
filtervariants Filter variants across replicates
consensus Call consensus from aligned BAM file
getmasked Detect primer mismatches and get primer indices for the amplicon to be masked
removereads Remove reads from trimmed BAM file
version Show version information
To view detailed usage for each command, type ivar <command>, for example:
[user@cn4471 ~]$ ivar trim -h
Usage: ivar trim -i <input.bam> -b <primers.bed> -p <prefix> [-m <min-length>] [-q <min-quality>] [-s <sliding-window-width>]
Input Options Description
-i (Required) Sorted bam file, with aligned reads, to trim primers and quality
-b (Required) BED file with primer sequences and positions
-m Minimum length of read to retain after trimming (Default: 30)
-q Minimum quality threshold for sliding window to pass (Default: 20)
-s Width of sliding window (Default: 4)
-e Include reads with no primers. By default, reads with no primers are excluded
Output Options Description
-p (Required) Prefix for the output BAM file
Perform the trimming on sample data:
[user@cn4471 ~]$ cp test_isize.bed test_primers.bed [user@cn4471 ~]$ ivar trim -b test_primers.bed -p test.trimmed -i test.bam -q 15 -m 50 -s 4 Found 218 primers in BED file Amplicons detected: Number of references in file: 1 NC_045512.2 Using Region: NC_045512.2 Found 8 mapped reads Found 0 unmapped reads Sorted By Coordinate ------- Processed 10% reads ... Processed 20% reads ... Processed 30% reads ... Processed 40% reads ... ------- Results: Primer Name Read Count nCoV-2019_1_LEFT 0 nCoV-2019_1_RIGHT 0 nCoV-2019_2_LEFT 0 nCoV-2019_2_RIGHT 0 nCoV-2019_3_LEFT 0 nCoV-2019_3_RIGHT 0 nCoV-2019_4_LEFT 0 nCoV-2019_4_RIGHT 0 ... nCoV-2019_96_LEFT 0 nCoV-2019_96_RIGHT 0 nCoV-2019_97_LEFT 0 nCoV-2019_97_RIGHT 0 nCoV-2019_98_LEFT 0 nCoV-2019_98_RIGHT 0 Trimmed primers from 100% (8) of reads. 0% (0) of reads were quality trimmed below the minimum length of 50 bp and were not written to file. 0% (0) of reads that started outside of primer regions were not written to file 50% (4) of reads had their insert size smaller than their read lengthEnd the interactive session:
[user@cn4471 ~]$ exit salloc.exe: Relinquishing job allocation 46116226 [user@biowulf ~]$