Workflow fix

.github/workflows/ccpp.yml

@@ -13,19 +13,19 @@ jobs:
 
     strategy:
       matrix:
-        os: [ubuntu-18.04, ubuntu-20.04]
+        os: [ubuntu-22.04]
 
     steps:
     - uses: actions/checkout@master
     - name: download htslib
       run: |
         wget https://github.com/samtools/htslib/archive/1.10.2.tar.gz
-        sudo apt-get install gcc-7
+        sudo apt-get install gcc-11
     - name: untar
       run: tar xf 1.10.2.tar.gz
     - name: install htslib
       env:
-        CC: gcc-7
+        CC: gcc-11
       run: |
         cd htslib-1.10.2/
         autoheader
@@ -36,47 +36,42 @@ jobs:
         sudo ldconfig
     - name: autotools
       env:
-        CC: gcc-7
+        CC: gcc-11
       run: ./autogen.sh
     - name: configure
       env:
-        CC: gcc-7
+        CC: gcc-11
       run: ./configure
     - name: make
       env:
-        CC: gcc-7
+        CC: gcc-11
       run: make
     - name: make check
       env:
-        CC: gcc-7
+        CC: gcc-11
       run: make check
     - name: make install
       env:
-        CC: gcc-7
+        CC: gcc-11
       run: sudo make install
 
   build-macos:
 
-    runs-on: [ macos-11 ]
+    runs-on: [ macos-14 ]
 
     steps:
     - uses: actions/checkout@master
     - name: install GNU autotools
       run: |
-        curl -O http://ftp.gnu.org/gnu/autoconf/autoconf-2.69.tar.gz
-        tar zxvf autoconf-2.69.tar.gz
-        cd autoconf-2.69
-        ./configure && make && sudo make install
-        brew install automake libtool
+        brew install autoconf automake libtool
     - name: download htslib
-      run: wget https://github.com/samtools/htslib/archive/1.10.2.tar.gz
+      run: wget https://github.com/samtools/htslib/releases/download/1.12/htslib-1.12.tar.bz2
     - name: untar
-      run: tar xf 1.10.2.tar.gz
+      run: tar xf htslib-1.12.tar.bz2
     - name: install htslib
       run: |
-        cd htslib-1.10.2/
-        autoheader
-        autoconf
+        cd htslib-1.12/
+        autoreconf -i
         ./configure
         make
         sudo make install

Dockerfile

@@ -1,18 +1,18 @@
-FROM ubuntu:18.04
+FROM ubuntu:20.04
 MAINTAINER Karthik G <gkarthik@scripps.edu>
 
 RUN apt-get update
 RUN apt-get install -y build-essential autoconf zlib1g-dev python3 wget libbz2-dev liblzma-dev libncurses-dev git bedtools python3-pip vim nano
 # HTSlib
 RUN cd root/ &&\
-    wget https://github.com/samtools/htslib/releases/download/1.9/htslib-1.9.tar.bz2 &&\
-    tar xvf htslib-1.9.tar.bz2 &&\
-    cd htslib-1.9/ &&\
+    wget https://github.com/samtools/htslib/releases/download/1.10.2/htslib-1.10.2.tar.bz2 &&\
+    tar xvf htslib-1.10.2.tar.bz2 &&\
+    cd htslib-1.10.2/ &&\
     ./configure &&\
     make &&\
     make install &&\
     cd ../ &&\
-    rm htslib-1.9.tar.bz2
+    rm htslib-1.10.2.tar.bz2
 ENV LD_LIBRARY_PATH /usr/local/lib:$LD_LIBRARY_PATH
 # SAMtools
 RUN cd root &&\
@@ -34,12 +34,12 @@ RUN cd root/ &&\
     make install
 # bwa
 RUN cd root/ &&\
-    wget https://github.com/lh3/bwa/archive/v0.7.17.tar.gz &&\
-    tar xvf v0.7.17.tar.gz &&\
-    cd bwa-0.7.17/ &&\
+    wget https://github.com/lh3/bwa/archive/v0.7.18.tar.gz &&\
+    tar xvf v0.7.18.tar.gz &&\
+    cd bwa-0.7.18/ &&\
     make &&\
     cd ../ &&\
-    rm v0.7.17.tar.gz
-ENV PATH /root/bwa-0.7.17:$PATH
+    rm v0.7.18.tar.gz
+ENV PATH /root/bwa-0.7.18:$PATH
 # Snakemake
 RUN pip3 install pandas snakemake

Doxyfile

@@ -58,7 +58,7 @@ PROJECT_LOGO           =
 # entered, it will be relative to the location where doxygen was started. If
 # left blank the current directory will be used.
 
-OUTPUT_DIRECTORY       = /Users/karthik/Documents/code/ivar/docs
+OUTPUT_DIRECTORY       = ./docs
 
 # If the CREATE_SUBDIRS tag is set to YES then doxygen will create 4096 sub-
 # directories (in 2 levels) under the output directory of each output format and

README.md

@@ -18,7 +18,7 @@ bioRxiv doi: [https://doi.org/10.1101/383513](https://doi.org/10.1101/383513)
 
 Manual for iVar is available [here](https://andersen-lab.github.io/ivar/html/).
 
-## Insallation
+## Installation
 
 ### Dependencies
 

config.h.in

@@ -3,26 +3,23 @@
 /* Define to 1 if you have the <inttypes.h> header file. */
 #undef HAVE_INTTYPES_H
 
-/* Define to 1 if you have the `hts' library (-lhts). */
+/* Define to 1 if you have the 'hts' library (-lhts). */
 #undef HAVE_LIBHTS
 
-/* Define to 1 if you have the `pthread' library (-lpthread). */
+/* Define to 1 if you have the 'pthread' library (-lpthread). */
 #undef HAVE_LIBPTHREAD
 
-/* Define to 1 if you have the `z' library (-lz). */
+/* Define to 1 if you have the 'z' library (-lz). */
 #undef HAVE_LIBZ
 
-/* Define to 1 if your system has a GNU libc compatible `malloc' function, and
+/* Define to 1 if your system has a GNU libc compatible 'malloc' function, and
    to 0 otherwise. */
 #undef HAVE_MALLOC
 
-/* Define to 1 if you have the `memmove' function. */
+/* Define to 1 if you have the 'memmove' function. */
 #undef HAVE_MEMMOVE
 
-/* Define to 1 if you have the <memory.h> header file. */
-#undef HAVE_MEMORY_H
-
-/* Define to 1 if your system has a GNU libc compatible `realloc' function,
+/* Define to 1 if your system has a GNU libc compatible 'realloc' function,
    and to 0 otherwise. */
 #undef HAVE_REALLOC
 
@@ -32,6 +29,9 @@
 /* Define to 1 if you have the <stdint.h> header file. */
 #undef HAVE_STDINT_H
 
+/* Define to 1 if you have the <stdio.h> header file. */
+#undef HAVE_STDIO_H
+
 /* Define to 1 if you have the <stdlib.h> header file. */
 #undef HAVE_STDLIB_H
 
@@ -50,7 +50,7 @@
 /* Define to 1 if you have the <unistd.h> header file. */
 #undef HAVE_UNISTD_H
 
-/* Define to 1 if the system has the type `_Bool'. */
+/* Define to 1 if the system has the type '_Bool'. */
 #undef HAVE__BOOL
 
 /* Name of package */
@@ -74,7 +74,9 @@
 /* Define to the version of this package. */
 #undef PACKAGE_VERSION
 
-/* Define to 1 if you have the ANSI C header files. */
+/* Define to 1 if all of the C89 standard headers exist (not just the ones
+   required in a freestanding environment). This macro is provided for
+   backward compatibility; new code need not use it. */
 #undef STDC_HEADERS
 
 /* Version number of package */
@@ -90,7 +92,7 @@
    #define below would cause a syntax error. */
 #undef _UINT8_T
 
-/* Define to `__inline__' or `__inline' if that's what the C compiler
+/* Define to '__inline__' or '__inline' if that's what the C compiler
    calls it, or to nothing if 'inline' is not supported under any name.  */
 #ifndef __cplusplus
 #undef inline

configure.ac

@@ -2,7 +2,7 @@
 # Process this file with autoconf to produce a configure script.
 
 AC_PREREQ([2.63])
-AC_INIT([ivar], [1.3.1], [gkarthik@scripps.edu])
+AC_INIT([ivar], [1.4.4], [gkarthik@scripps.edu])
 AM_INIT_AUTOMAKE([foreign subdir-objects])
 AC_CONFIG_HEADERS([config.h])
 

data/test.strand.pileup

@@ -0,0 +1,20 @@
+test	1	G	20	GGGGGGGGGGGGGGGGGGGG	????????????????????
+test	2	A	20	AAAAAAAAAAAAAAAAAAAA	????????????????????
+test	3	G	20	GGGGGGGGGGGGGGGGGGGG	????????????????????
+test	4	G	20	GGGGGGGGGGGGGGGGGGGG	????????????????????
+test	5	C	20	CCCCCCCCCCTTTTTTTTTT	????????????????????
+test	6	T	20	TTTTTTTTTTTTTTTTTTTT	????????????????????
+test	7	G	20	GGGGGGGGGGGGGGGGGGGG	????????????????????
+test	8	C	20	CCCCCCCCCCCCCCCCCCCC	????????????????????
+test	9	C	20	CCCCCCCCCCCCCCCCCCCC	????????????????????
+test	10	A	20	AAAAAAAAAAAAAAAAAAAA	????????????????????
+test	11	G	20	GGGGGGGGGGGGGGGGGGGG	????????????????????
+test	12	C	20	CCCCCCCCCCCCCCCCCCCC	????????????????????
+test	13	C	20	CCCCCCCCCCCCCCCCCCCC	????????????????????
+test	14	G	20	GGGGGGGGGGGGGGGGGGGG	????????????????????
+test	15	G	20	GGGGGGGGGGCCCCCCCCCC	????????????????????
+test	16	A	20	AAAAAAAAAAAAAAAAAAAA	????????????????????
+test	17	C	20	CCCCCCCCCCCCCCCCCCCC	????????????????????
+test	18	T	20	TTTTTTTTTTTTTTTTTTTT	????????????????????
+test	19	T	20	TTTTTTTTTTTTTTTTTTTT	????????????????????
+test	20	C	20	CCCCCCCCCCCCCCCCCCCC	????????????????????

data/test_strand.gff

@@ -0,0 +1,3 @@
+## GFF3 file format
+test	Genbank	CDS	1	9	.	+	.	ID=test1;Note=positiveStrand;gene=A;
+test	Genbank	CDS	11	20	.	-	.	ID=test2;Note=negativeStrand;gene=B;

docs/MANUAL.md

@@ -42,31 +42,39 @@ Command:
 ```
 ivar trim
 
-Usage: ivar trim -i <input.bam> -b <primers.bed> -p <prefix> [-m <min-length>] [-q <min-quality>] [-s <sliding-window-width>]
+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
+           -i    Sorted bam file, with aligned reads, to trim primers and quality. If not specified will read from standard in
            -b    (Required) BED file with primer sequences and positions
            -f    Primer pair information file containing left and right primer names for the same amplicon separated by a tab
                  If provided, reads will be filtered based on their overlap with amplicons prior to trimming
-           -m    Minimum length of read to retain after trimming (Default: 30)
+           -m    Minimum length of read to retain after trimming (Default: 50% the average length of the first 1000 reads)
            -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
            -k    Keep reads to allow for reanalysis: keep reads which would be dropped by
                  alignment length filter or primer requirements, but mark them QCFAIL
 
 Output Options   Description
-           -p    (Required) Prefix for the output BAM file
+           -p    Prefix for the output BAM file. If none is specified the output will write to standard out.
 ```
 
 Example Usage:
 ```
 ivar trim -b test_primers.bed -p test.trimmed -i test.bam -q 15 -m 50 -s 4
+samtools view -h test.bam | ivar trim -b test_primers.bed -p test.trimmed 
 ```
 
 The command above will produce a trimmed BAM file test.trimmed.bam after trimming the aligned reads in test.bam using the primer positions specified in test_primers.bed and a minimum quality threshold of **15**, minimum read length of **50** and a sliding window of **4**.
 
+Example Usage:
+```
+bwa mem -t 32 reference.fa 1.fq 2.fq | ivar trim -b test_primers.bed -x 3 -m 30 | samtools sort - | samtools mpileup -aa -A -Q 0 -d 0 - | ivar consensus -p test_consensus -m 10 -n N -t 0.5
+```
+
+The command above will allow you to go from alignment to consensus sequence in a single command using the bwa aligner.
+
 Example BED file
 
 ```
@@ -112,6 +120,7 @@ Input Options    Description
            -q    Minimum quality score threshold to count base (Default: 20)
            -t    Minimum frequency threshold(0 - 1) to call variants (Default: 0.03)
            -m    Minimum read depth to call variants (Default: 0)
+           -G    Count gaps towards depth. By default, gaps are not counted           
            -r    Reference file used for alignment. This is used to translate the nucleotide sequences and identify intra host single nucleotide variants
            -g    A GFF file in the GFF3 format can be supplied to specify coordinates of open reading frames (ORFs). In absence of GFF file, amino acid translation will not be done.
 
@@ -280,6 +289,7 @@ Note : samtools mpileup output must be piped into ivar consensus
 Input Options    Description
            -q    Minimum quality score threshold to count base (Default: 20)
            -t    Minimum frequency threshold(0 - 1) to call consensus. (Default: 0)
+           -c    Minimum insertion frequency threshold(0 - 1) to call consensus. (Default: 0.8)
                  Frequently used thresholds | Description
                  ---------------------------|------------
                                           0 | Majority or most common base