From 7696766a73df1914e58614ebe0c48cfe9af6a0ff Mon Sep 17 00:00:00 2001
From: Alaa Abdel Latif <al.a.latif.94@gmail.com>
Date: Thu, 4 Feb 2021 19:11:36 -0800
Subject: [PATCH 1/4] added optional -x flag for ivar trim. allows trimming of
 reads that occur at a specified offset position relative to the primer
 sequence positions

---
 configure.ac                          |  2 +-
 src/ivar.cpp                          | 12 +++++++++---
 src/primer_bed.cpp                    |  6 +++---
 src/primer_bed.h                      |  2 +-
 src/trim_primer_quality.cpp           |  4 ++--
 src/trim_primer_quality.h             |  2 +-
 tests/test_amplicon_search.cpp        |  7 ++++---
 tests/test_isize_trim.cpp             |  3 ++-
 tests/test_primer_bed.cpp             |  3 ++-
 tests/test_primer_trim.cpp            |  3 ++-
 tests/test_primer_trim_edge_cases.cpp |  3 ++-
 tests/test_trim.cpp                   |  3 ++-
 tests/test_unpaired_trim.cpp          |  3 ++-
 13 files changed, 33 insertions(+), 20 deletions(-)

diff --git a/configure.ac b/configure.ac
index ad994ac1..c4eb867e 100644
--- a/configure.ac
+++ b/configure.ac
@@ -2,7 +2,7 @@
 # Process this file with autoconf to produce a configure script.
 
 AC_PREREQ([2.63])
-AC_INIT([ivar], [1.3], [gkarthik@scripps.edu])
+AC_INIT([ivar], [1.3.1], [gkarthik@scripps.edu])
 AM_INIT_AUTOMAKE([foreign subdir-objects])
 AC_CONFIG_HEADERS([config.h])
 
diff --git a/src/ivar.cpp b/src/ivar.cpp
index 161f4c02..0ef226c1 100755
--- a/src/ivar.cpp
+++ b/src/ivar.cpp
@@ -16,7 +16,7 @@
 #include "suffix_tree.h"
 #include "get_common_variants.h"
 
-const std::string VERSION = "1.3";
+const std::string VERSION = "1.3.1";
 
 struct args_t {
   std::string bam;		// -i
@@ -37,6 +37,7 @@ struct args_t {
   char gap;			// -n
   bool keep_min_coverage;	// -k
   std::string primer_pair_file;	// -f
+  int32_t primer_offset; // -x
   std::string file_list;		// -f
   bool write_no_primers_flag;	// -e
   std::string gff;		// -g
@@ -67,6 +68,7 @@ void print_trim_usage(){
     "           -b    BED file with primer sequences and positions. If no BED file is specified, only quality trimming will be done.\n"
     "           -f    [EXPERIMENTAL] Primer pair information file containing left and right primer names for the same amplicon separated by a tab\n"
     "                 If provided, reads that do not fall within atleat one amplicon will be ignored prior to primer trimming.\n"
+    "           -x    Primer position offset (Default: 0). Reads that occur at the specified offset positions relative to primer positions will also be trimmed.\n"
     "           -m    Minimum length of read to retain after trimming (Default: 30)\n"
     "           -q    Minimum quality threshold for sliding window to pass (Default: 20)\n"
     "           -s    Width of sliding window (Default: 4)\n"
@@ -165,7 +167,7 @@ void print_version_info(){
     "\nPlease raise issues and bug reports at https://github.com/andersen-lab/ivar/\n\n";
 }
 
-static const char *trim_opt_str = "i:b:f:p:m:q:s:ekh?";
+static const char *trim_opt_str = "i:b:f:x:p:m:q:s:ekh?";
 static const char *variants_opt_str = "p:t:q:m:r:g:h?";
 static const char *consensus_opt_str = "i:p:q:t:m:n:kh?";
 static const char *removereads_opt_str = "i:p:t:b:h?";
@@ -221,6 +223,7 @@ int main(int argc, char* argv[]){
     g_args.keep_for_reanalysis = false;
     g_args.bed = "";
     g_args.primer_pair_file = "";
+    g_args.primer_offset = 0;
     opt = getopt( argc, argv, trim_opt_str);
     while( opt != -1 ) {
       switch( opt ) {
@@ -232,6 +235,9 @@ int main(int argc, char* argv[]){
 	break;
       case 'f':
   g_args.primer_pair_file = optarg;
+  break;
+      case 'x':
+  g_args.primer_offset = std::stoi(optarg);
   break;
       case 'p':
 	g_args.prefix = optarg;
@@ -264,7 +270,7 @@ int main(int argc, char* argv[]){
       return -1;
     }
     g_args.prefix = get_filename_without_extension(g_args.prefix,".bam");
-    res = trim_bam_qual_primer(g_args.bam, g_args.bed, g_args.prefix, g_args.region, g_args.min_qual, g_args.sliding_window, cl_cmd.str(), g_args.write_no_primers_flag, g_args.keep_for_reanalysis, g_args.min_length, g_args.primer_pair_file);
+    res = trim_bam_qual_primer(g_args.bam, g_args.bed, g_args.prefix, g_args.region, g_args.min_qual, g_args.sliding_window, cl_cmd.str(), g_args.write_no_primers_flag, g_args.keep_for_reanalysis, g_args.min_length, g_args.primer_pair_file, g_args.primer_offset);
   }
   // ivar variants
   else if (cmd.compare("variants") == 0){
diff --git a/src/primer_bed.cpp b/src/primer_bed.cpp
index 16e38cee..4b38f9f6 100644
--- a/src/primer_bed.cpp
+++ b/src/primer_bed.cpp
@@ -85,7 +85,7 @@ void print_bed_format(){
   std::cout << "It requires the following columns delimited by a tab: chrom, chromStart, chromEnd, name, score, strand" << std::endl;
 }
 
-std::vector<primer> populate_from_file(std::string path){
+std::vector<primer> populate_from_file(std::string path, int32_t offset = 0){
   std::ifstream  data(path.c_str());
   std::string line;
   std::vector<primer> primers;
@@ -103,7 +103,7 @@ std::vector<primer> populate_from_file(std::string path){
 	break;
       case 1:
 	if(std::all_of(cell.begin(), cell.end(), ::isdigit)) {
-	  p.set_start(std::stoul(cell));
+	  p.set_start(std::stoul(cell) - offset);
 	} else {
 	  print_bed_format();
 	  primers.clear();
@@ -112,7 +112,7 @@ std::vector<primer> populate_from_file(std::string path){
 	break;
       case 2:
 	if(std::all_of(cell.begin(), cell.end(), ::isdigit)) {
-	  p.set_end(std::stoul(cell)-1); // Bed format - End is not 0 based
+	  p.set_end(std::stoul(cell) - 1 + offset); // Bed format - End is not 0 based
 	} else {
 	  print_bed_format();
 	  primers.clear();
diff --git a/src/primer_bed.h b/src/primer_bed.h
index 2ff284ec..4e064085 100644
--- a/src/primer_bed.h
+++ b/src/primer_bed.h
@@ -46,7 +46,7 @@ class primer {
 
 };
 
-std::vector<primer> populate_from_file(std::string path);
+std::vector<primer> populate_from_file(std::string path, int32_t offset);
 std::vector<primer> get_primers(std::vector<primer> p, unsigned int pos);
 int get_primer_indice(std::vector<primer> p, std::string name);
 int populate_pair_indices(std::vector<primer> &primers, std::string path);
diff --git a/src/trim_primer_quality.cpp b/src/trim_primer_quality.cpp
index 38db3d9d..5d73b5a0 100755
--- a/src/trim_primer_quality.cpp
+++ b/src/trim_primer_quality.cpp
@@ -346,12 +346,12 @@ bool amplicon_filter(IntervalTree amplicons, bam1_t* r){
   return amplicon_flag;
 }
 
-int trim_bam_qual_primer(std::string bam, std::string bed, std::string bam_out, std::string region_, uint8_t min_qual, uint8_t sliding_window, std::string cmd, bool write_no_primer_reads, bool keep_for_reanalysis, int min_length = 30, std::string pair_info = "") {
+int trim_bam_qual_primer(std::string bam, std::string bed, std::string bam_out, std::string region_, uint8_t min_qual, uint8_t sliding_window, std::string cmd, bool write_no_primer_reads, bool keep_for_reanalysis, int min_length = 30, std::string pair_info = "", int32_t primer_offset = 0) {
   int retval = 0;
   std::vector<primer> primers;
   int max_primer_len = 0;
   if(!bed.empty()){
-    primers = populate_from_file(bed);
+    primers = populate_from_file(bed, primer_offset);
     if(primers.size() == 0){
       std::cout << "Exiting." << std::endl;
       return -1;
diff --git a/src/trim_primer_quality.h b/src/trim_primer_quality.h
index dd08ba0b..3fe1f82b 100755
--- a/src/trim_primer_quality.h
+++ b/src/trim_primer_quality.h
@@ -31,7 +31,7 @@ inline void free_cigar(cigar_ t) { if (t.free_cig) free(t.cigar); }
 void add_pg_line_to_header(bam_hdr_t** hdr, char *cmd);
 
 
-int trim_bam_qual_primer(std::string bam, std::string bed, std::string bam_out, std::string region_, uint8_t min_qual, uint8_t sliding_window, std::string cmd, bool write_no_primer_reads, bool mark_qcfail_flag, int min_length, std::string pair_info);
+int trim_bam_qual_primer(std::string bam, std::string bed, std::string bam_out, std::string region_, uint8_t min_qual, uint8_t sliding_window, std::string cmd, bool write_no_primer_reads, bool mark_qcfail_flag, int min_length, std::string pair_info, int32_t primer_offset);
 void free_cigar(cigar_ t);
 int32_t get_pos_on_query(uint32_t *cigar, uint32_t ncigar, int32_t pos, int32_t ref_start);
 int32_t get_pos_on_reference(uint32_t *cigar, uint32_t ncigar, uint32_t pos, uint32_t ref_start);
diff --git a/tests/test_amplicon_search.cpp b/tests/test_amplicon_search.cpp
index 5bd705a6..ef4fba9a 100755
--- a/tests/test_amplicon_search.cpp
+++ b/tests/test_amplicon_search.cpp
@@ -5,11 +5,11 @@
 #include "../src/primer_bed.h"
 #include "../src/interval_tree.h"
 
-int test_amplicon_search(std::string bam_file, std::string bed_file, std::string pair_info_file, bool expected[])
+int test_amplicon_search(std::string bam_file, std::string bed_file, std::string pair_info_file, int32_t primer_offset, bool expected[])
 {
   std::vector<primer> primers;
   IntervalTree amplicons;
-  primers = populate_from_file(bed_file);
+  primers = populate_from_file(bed_file, primer_offset);
   std::cout << "Total Number of primers: " << primers.size() << std::endl;
   amplicons = populate_amplicons(pair_info_file, primers);
   samFile *in = hts_open(bam_file.c_str(), "r");
@@ -35,13 +35,14 @@ int main(){
   int success;
   std::string bam = "../data/test_amplicon.sorted.bam";
   std::string pair_indice = "../data/pair_info_2.tsv";
+  int32_t primer_offset = 0;
   std::string bed = "../data/test_isize.bed";
   std::string prefix = "/tmp/data/trim_amplicon";
   std::string bam_out = "/tmp/trim_amplicon.bam";
 
   IntervalTree amplicons;
   bool expected[8] = {true, false, true, false, true, false, true, false};
-  success = test_amplicon_search(bam, bed, pair_indice, expected);
+  success = test_amplicon_search(bam, bed, pair_indice, primer_offset, expected);
   amplicons.inOrder();
   return success;
 }
diff --git a/tests/test_isize_trim.cpp b/tests/test_isize_trim.cpp
index 6deaf1fa..bfdd39bb 100755
--- a/tests/test_isize_trim.cpp
+++ b/tests/test_isize_trim.cpp
@@ -21,6 +21,7 @@ int test_isize_trim(uint8_t min_qual, uint8_t sliding_window, bool no_write_flag
     std::string bam = "../data/test_isize.sorted.bam";
     std::string bed = "../data/test_isize.bed";
     std::string pair_info = "";
+    int32_t primer_offset = 0;
     std::string prefix = "../data/trim_isize";
     std::string bam_out = "../data/trim_isize.bam";
 
@@ -29,7 +30,7 @@ int test_isize_trim(uint8_t min_qual, uint8_t sliding_window, bool no_write_flag
     std::string cmd = "@PG\tID:ivar-trim\tPN:ivar\tVN:1.0.0\tCL:ivar trim\n";
 
     // Test and check result
-    res = trim_bam_qual_primer(bam, bed, prefix, region_, min_qual, sliding_window, cmd, no_write_flag, keep_for_reanalysis, min_length, pair_info);
+    res = trim_bam_qual_primer(bam, bed, prefix, region_, min_qual, sliding_window, cmd, no_write_flag, keep_for_reanalysis, min_length, pair_info, primer_offset);
 
     if (res) {
         success = -1;
diff --git a/tests/test_primer_bed.cpp b/tests/test_primer_bed.cpp
index f395a8e0..9581107a 100755
--- a/tests/test_primer_bed.cpp
+++ b/tests/test_primer_bed.cpp
@@ -7,7 +7,8 @@ int main(){
   int num_tests = 2;
   int success = 0;
   std::vector<primer>::iterator it;
-  std::vector<primer> primers = populate_from_file("../data/test.bed");
+  int32_t primer_offset = 0;
+  std::vector<primer> primers = populate_from_file("../data/test.bed", primer_offset);
   std::string primer_names[] = {"WNV_400_1_LEFT", "WNV_400_1_LEFT_alt", "WNV_400_2_LEFT", "WNV_400_1_RIGHT", "WNV_400_2_RIGHT", "WNV_400_3_LEFT", "WNV_400_2_LEFT_alt", "WNV_400_2_RIGHT_alt"};
   int primer_indices[] = {0,1,2,3,4,5,6,7,8};
   unsigned int primer_start[] = {8,7, 230, 359,658,569,251,352};
diff --git a/tests/test_primer_trim.cpp b/tests/test_primer_trim.cpp
index 4bede7cf..02357fcb 100755
--- a/tests/test_primer_trim.cpp
+++ b/tests/test_primer_trim.cpp
@@ -7,7 +7,8 @@
 int main(){
   int success = 0;
   std::string bam = "../data/test.unmapped.sorted.bam";
-  std::vector<primer> primers = populate_from_file("../data/test.bed");
+  int32_t primer_offset = 0;
+  std::vector<primer> primers = populate_from_file("../data/test.bed", primer_offset);
   int max_primer_len = 0;
   max_primer_len = get_bigger_primer(primers);
   std::string region_;
diff --git a/tests/test_primer_trim_edge_cases.cpp b/tests/test_primer_trim_edge_cases.cpp
index a1da782b..f9f948fa 100755
--- a/tests/test_primer_trim_edge_cases.cpp
+++ b/tests/test_primer_trim_edge_cases.cpp
@@ -4,7 +4,8 @@
 int main(){
   int success = 0;
   std::string bam = "../data/primer_only/primer_edge_cases.bam";
-  std::vector<primer> primers = populate_from_file("../data/test.bed");
+  int32_t primer_offset = 0;
+  std::vector<primer> primers = populate_from_file("../data/test.bed", primer_offset);
   int max_primer_len = 0;
   max_primer_len = get_bigger_primer(primers);
   std::string region_;
diff --git a/tests/test_trim.cpp b/tests/test_trim.cpp
index e0673557..a222a3cd 100755
--- a/tests/test_trim.cpp
+++ b/tests/test_trim.cpp
@@ -21,6 +21,7 @@ int test_trim(uint8_t min_qual, uint8_t sliding_window, bool no_write_flag, bool
   std::string bam = "../data/test.unmapped.sorted.bam";
   std::string bed = "../data/test.bed";
   std::string pair_info = "";
+  int32_t primer_offset = 0;
   std::string prefix = "/tmp/trim";
   std::string bam_out = "/tmp/trim.bam";
 
@@ -28,7 +29,7 @@ int test_trim(uint8_t min_qual, uint8_t sliding_window, bool no_write_flag, bool
   std::string cmd = "@PG\tID:ivar-trim\tPN:ivar\tVN:1.0.0\tCL:ivar trim\n";
 
   // Test and check result
-  res = trim_bam_qual_primer(bam, bed, prefix, region_, min_qual, sliding_window, cmd, no_write_flag, keep_for_reanalysis, min_length, pair_info);
+  res = trim_bam_qual_primer(bam, bed, prefix, region_, min_qual, sliding_window, cmd, no_write_flag, keep_for_reanalysis, min_length, pair_info, primer_offset);
   if (res) {
     success = -1;
     std::cerr << testname << " failed: trim_bam_qual_primer() returned " << res << std::endl;
diff --git a/tests/test_unpaired_trim.cpp b/tests/test_unpaired_trim.cpp
index c4451167..5260d92e 100755
--- a/tests/test_unpaired_trim.cpp
+++ b/tests/test_unpaired_trim.cpp
@@ -7,7 +7,8 @@
 int main(){
   int success = 0;
   std::string bam = "../data/test.sim.merged.sorted.bam";
-  std::vector<primer> primers = populate_from_file("../data/test_merged.bed");
+  int32_t primer_offset = 0;
+  std::vector<primer> primers = populate_from_file("../data/test_merged.bed", primer_offset);
   int max_primer_len = 0;
   max_primer_len = get_bigger_primer(primers);
   std::string region_;

From 0ff381aa3b2bb4da73b5f045a5f9b7feafa98023 Mon Sep 17 00:00:00 2001
From: Alaa Abdel Latif <al.a.latif.94@gmail.com>
Date: Thu, 4 Feb 2021 19:26:22 -0800
Subject: [PATCH 2/4] add updates to these files to incorporate additional
 offset parameter for populate_from_file()

---
 src/remove_reads_from_amplicon.cpp | 3 ++-
 1 file changed, 2 insertions(+), 1 deletion(-)

diff --git a/src/remove_reads_from_amplicon.cpp b/src/remove_reads_from_amplicon.cpp
index 8d1863ad..79cd7d04 100644
--- a/src/remove_reads_from_amplicon.cpp
+++ b/src/remove_reads_from_amplicon.cpp
@@ -1,7 +1,8 @@
 #include "remove_reads_from_amplicon.h"
 
 int rmv_reads_from_amplicon(std::string bam, std::string region_, std::string bam_out, std::vector<std::string> amp, std::string bed, std::string cmd){
-  std::vector<primer> primers = populate_from_file(bed);
+  int32_t primer_offset = 0;
+  std::vector<primer> primers = populate_from_file(bed, primer_offset);
   if(primers.size() == 0){
     return 0;
   }

From 9b4ee076fb464cb2b72181fd63f76d7aafaac91b Mon Sep 17 00:00:00 2001
From: Alaa Abdel Latif <al.a.latif.94@gmail.com>
Date: Thu, 4 Feb 2021 19:29:32 -0800
Subject: [PATCH 3/4] add updates to these files to incorporate additional
 offset parameter for populate_from_file()

---
 src/get_masked_amplicons.cpp | 3 ++-
 1 file changed, 2 insertions(+), 1 deletion(-)

diff --git a/src/get_masked_amplicons.cpp b/src/get_masked_amplicons.cpp
index ac20a2bc..f10e6567 100644
--- a/src/get_masked_amplicons.cpp
+++ b/src/get_masked_amplicons.cpp
@@ -1,7 +1,8 @@
 #include "get_masked_amplicons.h"
 
 int get_primers_with_mismatches(std::string bed, std::string vpath, std::string out, std::string primer_pair_file){
-  std::vector<primer> primers = populate_from_file(bed);
+  int32_t primer_offset = 0;
+  std::vector<primer> primers = populate_from_file(bed, primer_offset);
   std::vector<primer> mismatches_primers;
   std::vector<primer> tmp;
   if(primers.size() == 0){

From 60e8e6bbde9c7620fa0379de75acfd1ffd38170b Mon Sep 17 00:00:00 2001
From: Alaa Abdel Latif <al.a.latif.94@gmail.com>
Date: Fri, 5 Feb 2021 09:16:14 -0800
Subject: [PATCH 4/4] overload populate_from_file()

---
 src/get_masked_amplicons.cpp       |  3 +-
 src/primer_bed.cpp                 | 69 ++++++++++++++++++++++++++++++
 src/primer_bed.h                   |  1 +
 src/remove_reads_from_amplicon.cpp |  3 +-
 tests/test_primer_bed.cpp          |  3 +-
 tests/test_primer_trim.cpp         |  3 +-
 tests/test_unpaired_trim.cpp       |  3 +-
 7 files changed, 75 insertions(+), 10 deletions(-)

diff --git a/src/get_masked_amplicons.cpp b/src/get_masked_amplicons.cpp
index f10e6567..ac20a2bc 100644
--- a/src/get_masked_amplicons.cpp
+++ b/src/get_masked_amplicons.cpp
@@ -1,8 +1,7 @@
 #include "get_masked_amplicons.h"
 
 int get_primers_with_mismatches(std::string bed, std::string vpath, std::string out, std::string primer_pair_file){
-  int32_t primer_offset = 0;
-  std::vector<primer> primers = populate_from_file(bed, primer_offset);
+  std::vector<primer> primers = populate_from_file(bed);
   std::vector<primer> mismatches_primers;
   std::vector<primer> tmp;
   if(primers.size() == 0){
diff --git a/src/primer_bed.cpp b/src/primer_bed.cpp
index 4b38f9f6..9fa99867 100644
--- a/src/primer_bed.cpp
+++ b/src/primer_bed.cpp
@@ -154,6 +154,75 @@ std::vector<primer> populate_from_file(std::string path, int32_t offset = 0){
   return primers;
 }
 
+std::vector<primer> populate_from_file(std::string path){
+  std::ifstream  data(path.c_str());
+  std::string line;
+  std::vector<primer> primers;
+  int16_t indice = 0;
+  while(std::getline(data,line)){ // Remove extra lineStream
+    std::stringstream lineStream(line);
+    std::string cell;
+    int ctr = 0;
+    primer p;
+    p.set_strand(0);		// Set strand to NULL
+    while(std::getline(lineStream,cell,'\t')){
+      switch(ctr){
+      case 0:
+	p.set_region(cell);
+	break;
+      case 1:
+	if(std::all_of(cell.begin(), cell.end(), ::isdigit)) {
+	  p.set_start(std::stoul(cell));
+	} else {
+	  print_bed_format();
+	  primers.clear();
+	  return primers;
+	}
+	break;
+      case 2:
+	if(std::all_of(cell.begin(), cell.end(), ::isdigit)) {
+	  p.set_end(std::stoul(cell) - 1); // Bed format - End is not 0 based
+	} else {
+	  print_bed_format();
+	  primers.clear();
+	  return primers;
+	}
+	break;
+      case 3:
+	p.set_name(cell);
+	break;
+      case 4:
+	if(std::all_of(cell.begin(), cell.end(), ::isdigit)) {
+	  p.set_score(stoi(cell));
+	} else {
+	  print_bed_format();  // score is missing, send warning but continue populating
+    std::cout << "\nWARNING: The BED file provided did not have the expected score column, but iVar will continue trimming\n" << std::endl;
+    p.set_score(-1);
+	}
+	break;
+      case 5:
+	if(cell[0] == '+' || cell[0] == '-')
+	  p.set_strand(cell[0]);
+	else {
+	  print_bed_format();
+	  primers.clear();
+	  return primers;
+	}
+      }
+      ctr++;
+    }
+    if(indice == 0 && ctr < 6)
+      std::cout << "Strand not found in primer BED file so strand will not be considered for trimming" << std::endl;
+    p.set_indice(indice);
+    p.set_pair_indice(-1);
+    p.set_read_count(0);
+    primers.push_back(p);
+    indice++;
+  }
+  std::cout << "Found " << primers.size() << " primers in BED file" << std::endl;
+  return primers;
+}
+
 std::vector<primer> get_primers(std::vector<primer> p, unsigned int pos){
   std::vector<primer> primers_with_mismatches;
   for(std::vector<primer>::iterator it = p.begin(); it != p.end(); ++it) {
diff --git a/src/primer_bed.h b/src/primer_bed.h
index 4e064085..c8aa71ca 100644
--- a/src/primer_bed.h
+++ b/src/primer_bed.h
@@ -47,6 +47,7 @@ class primer {
 };
 
 std::vector<primer> populate_from_file(std::string path, int32_t offset);
+std::vector<primer> populate_from_file(std::string path);
 std::vector<primer> get_primers(std::vector<primer> p, unsigned int pos);
 int get_primer_indice(std::vector<primer> p, std::string name);
 int populate_pair_indices(std::vector<primer> &primers, std::string path);
diff --git a/src/remove_reads_from_amplicon.cpp b/src/remove_reads_from_amplicon.cpp
index 79cd7d04..8d1863ad 100644
--- a/src/remove_reads_from_amplicon.cpp
+++ b/src/remove_reads_from_amplicon.cpp
@@ -1,8 +1,7 @@
 #include "remove_reads_from_amplicon.h"
 
 int rmv_reads_from_amplicon(std::string bam, std::string region_, std::string bam_out, std::vector<std::string> amp, std::string bed, std::string cmd){
-  int32_t primer_offset = 0;
-  std::vector<primer> primers = populate_from_file(bed, primer_offset);
+  std::vector<primer> primers = populate_from_file(bed);
   if(primers.size() == 0){
     return 0;
   }
diff --git a/tests/test_primer_bed.cpp b/tests/test_primer_bed.cpp
index 9581107a..f395a8e0 100755
--- a/tests/test_primer_bed.cpp
+++ b/tests/test_primer_bed.cpp
@@ -7,8 +7,7 @@ int main(){
   int num_tests = 2;
   int success = 0;
   std::vector<primer>::iterator it;
-  int32_t primer_offset = 0;
-  std::vector<primer> primers = populate_from_file("../data/test.bed", primer_offset);
+  std::vector<primer> primers = populate_from_file("../data/test.bed");
   std::string primer_names[] = {"WNV_400_1_LEFT", "WNV_400_1_LEFT_alt", "WNV_400_2_LEFT", "WNV_400_1_RIGHT", "WNV_400_2_RIGHT", "WNV_400_3_LEFT", "WNV_400_2_LEFT_alt", "WNV_400_2_RIGHT_alt"};
   int primer_indices[] = {0,1,2,3,4,5,6,7,8};
   unsigned int primer_start[] = {8,7, 230, 359,658,569,251,352};
diff --git a/tests/test_primer_trim.cpp b/tests/test_primer_trim.cpp
index 02357fcb..4bede7cf 100755
--- a/tests/test_primer_trim.cpp
+++ b/tests/test_primer_trim.cpp
@@ -7,8 +7,7 @@
 int main(){
   int success = 0;
   std::string bam = "../data/test.unmapped.sorted.bam";
-  int32_t primer_offset = 0;
-  std::vector<primer> primers = populate_from_file("../data/test.bed", primer_offset);
+  std::vector<primer> primers = populate_from_file("../data/test.bed");
   int max_primer_len = 0;
   max_primer_len = get_bigger_primer(primers);
   std::string region_;
diff --git a/tests/test_unpaired_trim.cpp b/tests/test_unpaired_trim.cpp
index 5260d92e..c4451167 100755
--- a/tests/test_unpaired_trim.cpp
+++ b/tests/test_unpaired_trim.cpp
@@ -7,8 +7,7 @@
 int main(){
   int success = 0;
   std::string bam = "../data/test.sim.merged.sorted.bam";
-  int32_t primer_offset = 0;
-  std::vector<primer> primers = populate_from_file("../data/test_merged.bed", primer_offset);
+  std::vector<primer> primers = populate_from_file("../data/test_merged.bed");
   int max_primer_len = 0;
   max_primer_len = get_bigger_primer(primers);
   std::string region_;