From 5b591e7a4515568f1b945339711c8c47306a89f6 Mon Sep 17 00:00:00 2001 From: Christophe Antoniewski Date: Sat, 27 Jan 2024 02:43:12 +0100 Subject: [PATCH 01/16] upgrade htseqcount --- tools/htseq_count/htseq-count.xml | 19 ++++++++---------- .../htseq-test-paired_othercounts.tsv | 1 + .../test-data/htseq-test_othercounts.tsv | 1 + .../test-data/htseq-test_samout.bam | Bin 67623 -> 66225 bytes 4 files changed, 10 insertions(+), 11 deletions(-) diff --git a/tools/htseq_count/htseq-count.xml b/tools/htseq_count/htseq-count.xml index 330e7a3fba6..973f82f78bb 100644 --- a/tools/htseq_count/htseq-count.xml +++ b/tools/htseq_count/htseq-count.xml @@ -4,8 +4,8 @@ htseq - htseq - samtools + htseq + samtools @@ -60,9 +60,12 @@ --format=sam '$name_sorted_alignment_filename' - "$gfffile" | awk '{if ($1 ~ "no_feature|ambiguous|too_low_aQual|not_aligned|alignment_not_unique") print $0 | "cat 1>&2"; else print $0}' + "$gfffile" | awk '{if ($1 ~ "no_feature|ambiguous|too_low_aQual|not_aligned|alignment_not_unique|aligned") print $0 | "cat 1>&2"; else print $0}' > '$counts' - 2> '$othercounts' + 2> '$othercounts' && + + echo -e "__aligned\t\$(cut -f2 '$counts' | awk '{s+=$1}END{print s}' -)" >> '$othercounts' && + sed -i 's/\"//g' '$counts' #if str( $advanced_options.advanced_options_selector ) == "advanced": #if $advanced_options.samout_conditional.samout == "Yes": @@ -155,7 +158,7 @@ - + @@ -166,12 +169,6 @@ - - - - - - diff --git a/tools/htseq_count/test-data/htseq-test-paired_othercounts.tsv b/tools/htseq_count/test-data/htseq-test-paired_othercounts.tsv index 2c8f02836cb..780515b12d9 100644 --- a/tools/htseq_count/test-data/htseq-test-paired_othercounts.tsv +++ b/tools/htseq_count/test-data/htseq-test-paired_othercounts.tsv @@ -3,3 +3,4 @@ __ambiguous 1 __too_low_aQual 68 __not_aligned 0 __alignment_not_unique 0 +__aligned 317 diff --git a/tools/htseq_count/test-data/htseq-test_othercounts.tsv b/tools/htseq_count/test-data/htseq-test_othercounts.tsv index 3c456a18fa3..3646b826444 100644 --- a/tools/htseq_count/test-data/htseq-test_othercounts.tsv +++ b/tools/htseq_count/test-data/htseq-test_othercounts.tsv @@ -3,3 +3,4 @@ __ambiguous 0 __too_low_aQual 0 __not_aligned 1336 __alignment_not_unique 0 +__aligned 343 diff --git a/tools/htseq_count/test-data/htseq-test_samout.bam b/tools/htseq_count/test-data/htseq-test_samout.bam index 38ab4ddde56ef902b3e815bcff683b9a5f0aa0c2..e12ab3ed7c52ade8b9d995adda574259751b8e5c 100644 GIT binary patch literal 66225 zcmV)6K*+xziwFb&00000{{{d;LjnMU0+o|LZ`3dt#$8%a(?V5X0EvO@$d@>F9OopY z7Airc-u2K`(M=rAzY=;Emn7u`BYXyCCKws{4h)@O;Y+Zx;y7(mI31{xExr2b@$)`@ zEkD@qE&W^uVC(UY=J&g(V`{^_ZYV?^r5P{dwXNY3ZP@QxaGHj>$h6&lmtw@ucyGTV z%yu0240#o?uxXi%%CDJYBGNF=QxGFck<-9Y#Rzs8v)jY9(xw(AxSXne&v9yP~4b@+z@l1!qV;z?IM1*{j z$4MvQa;3UaPLHbna+ZdNhezB<(n(wx*kDFtM7-ohT#hkIC<{;wBWhzvNMu6B0sW6d zgK;$Glk^~)##z~smO7lvV=c-flU{swQY7P4t>zlxg^jF#UVYKM#XNr@g7Hk`hiP^& 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2001 From: Christophe Antoniewski Date: Sat, 27 Jan 2024 02:51:17 +0100 Subject: [PATCH 02/16] commit htseq-test_samout.bam but variable output --- .../test-data/htseq-test_samout.bam | Bin 66225 -> 66225 bytes 1 file changed, 0 insertions(+), 0 deletions(-) diff --git a/tools/htseq_count/test-data/htseq-test_samout.bam b/tools/htseq_count/test-data/htseq-test_samout.bam index e12ab3ed7c52ade8b9d995adda574259751b8e5c..06cf22e7117d3b71854fd35fc98e57fdccab10de 100644 GIT binary patch delta 656 zcmV;B0&o4Xg#@vM1dtSeL2uJA7=}GI(6Yfa-~bW_az}1#$8nNMNE=LoNNZQrDe8&h zChiDp<0;7)>Bzu3T}PH5pU|p~TXsN3Y-S^=J9Pc4z6wG5}kT zcZ{&x#w}#@_u7$^S)8Q25NligCq}>9CUBZWnM{q{ZkyTIoAJ(nen~vXb^RF%Nc#^tV~kOU)~rM>b=2R; z^j_Dawrw|Q^f|DNU9S;V)F+gZrb}2s>LErhNK1&3>tXj|@{0P%r>rqJhnRTH&?@NI z*e{3Mdt?MV?VL}43n?eL(d#y~z9sCpi1Bc@rsm5;oS3kGY{J5v$cRreF>Z~y`V-SC zrbp!(I`UO?I6k$;$wcIqqdsoMyx_SghK|Fj&s+u_p9@HRKZ1TND8!hI1on{aj_j5a 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version --- tools/htseq_count/htseq-count.xml | 2 +- 1 file changed, 1 insertion(+), 1 deletion(-) diff --git a/tools/htseq_count/htseq-count.xml b/tools/htseq_count/htseq-count.xml index 973f82f78bb..cc16457a8c5 100644 --- a/tools/htseq_count/htseq-count.xml +++ b/tools/htseq_count/htseq-count.xml @@ -1,4 +1,4 @@ - + - Count aligned reads in a BAM file that overlap features in a GFF file htseq From c9e4e2321be1ae08272763c604baeb204d6a42f0 Mon Sep 17 00:00:00 2001 From: Christophe Antoniewski Date: Sat, 27 Jan 2024 03:26:37 +0100 Subject: [PATCH 04/16] update URLs --- tools/htseq_count/htseq-count.xml | 4 ++-- .../test-data/htseq-test_samout.bam | Bin 66225 -> 66225 bytes 2 files changed, 2 insertions(+), 2 deletions(-) diff --git a/tools/htseq_count/htseq-count.xml b/tools/htseq_count/htseq-count.xml index cc16457a8c5..62c00e53fe4 100644 --- a/tools/htseq_count/htseq-count.xml +++ b/tools/htseq_count/htseq-count.xml @@ -225,7 +225,7 @@ Overview This tool takes an alignment file in SAM or BAM format and feature file in GFF format and calculates the number of reads mapping to each feature. It uses the *htseq-count* script that is part of the HTSeq python module. See -http://www-huber.embl.de/users/anders/HTSeq/doc/count.html for details. +https://htseq.readthedocs.io/en/latest/ for details. A feature is an interval (i.e., a range of positions) on a chromosome or a union of such intervals. In the case of RNA-Seq, the features are typically genes, where @@ -377,7 +377,7 @@ Options: Show a usage summary and exit -.. _FAQ: https://htseq.readthedocs.io/en/release_0.9.1/count.html#frequenctly-asked-questions +.. _FAQ: https://htseq.readthedocs.io/en/master/index.html ]]> diff --git a/tools/htseq_count/test-data/htseq-test_samout.bam b/tools/htseq_count/test-data/htseq-test_samout.bam index 06cf22e7117d3b71854fd35fc98e57fdccab10de..92018cdfbcff1d5b6229607d2119d8ce1c9f9160 100644 GIT binary patch delta 656 zcmV;B0&o4Xg#@vM1dtSeKX2497{*;%P}4$HU;v4M?8uk+Pi*HTq!ubcq~7(=Rnbix zxx{Hh?~*D>sbGZ9z{~_ABj16c6D)iQR#qIRO$w)iD%sMjpB_K&ZKHkfeL#D9L45X8CK& zwrvYBs~%I)yx@fLn967ZIiFxiZD~U{q9J4?43NNf6cSXupqkk&0Dz(P*{=ql?*g!> zKyMe^-dO;DS5)bNjdzP0`X1Q2zXX;l=y~Al_a%+J5$Jun2Ciy_;@{pc>w`X!C(Bwf z@__$fMZ*!e8LVmx_5HxfuhrVZ18?@PYl~53apT@vtx+wXAD`BWe<;>YchE4U-%me@cABzYC000000RIMqj{=vE0s$h}yhWw} delta 656 zcmV;B0&o4Xg#@vM1dtSeL2uJA7=}GI(6Yfa-~bW_az}1#$8nNMNE=LoNNZQrDe8&h zChiDp<0;7)>Bzu3T}PH5pU|p~TXsN3Y-S^=J9Pc4z6wG5}kT zcZ{&x#w}#@_u7$^S)8Q25NligCq}>9CUBZWnM{q{ZkyTIoAJ(nen~vXb^RF%Nc#^tV~kOU)~rM>b=2R; z^j_Dawrw|Q^f|DNU9S;V)F+gZrb}2s>LErhNK1&3>tXj|@{0P%r>rqJhnRTH&?@NI z*e{3Mdt?MV?VL}43n?eL(d#y~z9sCpi1Bc@rsm5;oS3kGY{J5v$cRreF>Z~y`V-SC zrbp!(I`UO?I6k$;$wcIqqdsoMyx_SghK|Fj&s+u_p9@HRKZ1TND8!hI1on{aj_j5a z|Cz0Ye@oZv{VQoMv%-X_3HRrNsoI249v4T)1sqR{`~B^IjtQli@~Ie>8$~?iljIiL1QpX-%H08XD@)fR)&;@X{!N~2mnJvpxxf1hvaI#l(W51W;F q2)=);O69om>=yV12+SV{%me@cABzYC000000RIMqj{=vE0s$f<>O8Li From c4b10d8855dc7d1f9eeaf30a9fcf58e711efd5e4 Mon Sep 17 00:00:00 2001 From: Christophe Antoniewski Date: Sat, 27 Jan 2024 03:49:14 +0100 Subject: [PATCH 05/16] fix xml syntax of the tool --- tools/htseq_count/htseq-count.xml | 12 ++++++------ 1 file changed, 6 insertions(+), 6 deletions(-) diff --git a/tools/htseq_count/htseq-count.xml b/tools/htseq_count/htseq-count.xml index 62c00e53fe4..7be3ec85315 100644 --- a/tools/htseq_count/htseq-count.xml +++ b/tools/htseq_count/htseq-count.xml @@ -81,13 +81,13 @@ - + Mode to handle reads overlapping more than one feature. - + Specify whether the data is from a strand-specific assay. **Be sure to choose the correct value** (see help for more information). @@ -95,13 +95,13 @@ - + Skip all reads with alignment quality lower than the given minimum value. Feature type (3rd column in GFF file) to be used. All features of other types are ignored. The default, suitable for RNA-Seq and Ensembl GTF files, is exon. - + GFF attribute to be used as feature ID. Several GFF lines with the same feature ID will be considered as parts of the same feature. The feature ID is used to identity the counts in the output table. All features of the specified type MUST have a value for this attribute. The default, suitable for RNA-Seq and Ensembl GTF files, is gene_id. @@ -117,8 +117,8 @@ - - + + Write out all SAM alignment records into an output BAM file, annotating each line with its assignment to a feature or a special counter (as an optional field with tag ‘XF’). From c22cc710316a6458cd137faab2ad1920149d7384 Mon Sep 17 00:00:00 2001 From: Christophe Antoniewski Date: Sat, 27 Jan 2024 15:18:45 +0100 Subject: [PATCH 06/16] test on size of htseq-test_samout.bam --- tools/htseq_count/htseq-count.xml | 2 +- .../test-data/htseq-test_samout.bam | Bin 66225 -> 66227 bytes 2 files changed, 1 insertion(+), 1 deletion(-) diff --git a/tools/htseq_count/htseq-count.xml b/tools/htseq_count/htseq-count.xml index 7be3ec85315..15034d4d930 100644 --- a/tools/htseq_count/htseq-count.xml +++ b/tools/htseq_count/htseq-count.xml @@ -203,7 +203,7 @@ - + diff --git a/tools/htseq_count/test-data/htseq-test_samout.bam b/tools/htseq_count/test-data/htseq-test_samout.bam index 92018cdfbcff1d5b6229607d2119d8ce1c9f9160..8a5c05b20df0b76d9ea21d0f67b1a99709db6a1d 100644 GIT binary patch delta 662 zcmV;H0%`rRg#@#O1b-ik2m}BC000301^_}s0sw^qm6OkJ(=ZgrJvPv?!8G6i5(jce zZfqyDok~a>OoB*jSJWx$iQ@d|2(-zRc$?S}{szvR;K-5xfx}L4;h*5j6~|eVK~>PC zSbF;C``-Kdz4*~~cj@Oc09#LY48PaGEo2P#I$@UOQIZN7uYYX~o*9E)hrnqP=2>d& z_BzzYe8#)`CE;zx+!^vpLTp4(t@10z5O!+jc^cZ7Qp{>NESY1woVT0gO4+7_I(AJp zVwBUm@leYsrB2(aS&5j_R$n92xr{ruZ8vW8J+O@#Z-feH$P2|$JRKR#DzsIZXqiYQWUWqQI0T;T?~0h z0_eCrfB}y<6eNm57UKX%K}(7M_SV9Gxa;@-=d{RjX@A1hg!}WxRBS?+9m&HZ3CC0U zaIoDqVP>Xc8js4vI2wsba*$5rRJK$}6VCY|i~K03m!F*yDLZAWxkmQFM&zGYUv%$m zp1;b1(MguSPSS%>l;m+Jv;2*P5VA0}sxcMy1_cf5F%tr+Wkt{lSPX+ea7YMZ5yI+s z4Z?DRYJX<8004$MvsVp1-vwYs1L4oW8uSOAN~rH|}jz w8rAao$$6~!hhkI5zKY*|+^mZG;K!#bR24T~+y?=_0kGL=;Ddkymw*BRF#+2{!T)_0Uz(O&qzzX+!UlDoLqegwMdt1S2EgfuR#Dd`eGv3>+h;TS}&yZIUOl)j7D!*pjMzmp`ry)U%A>P1I#VB!waN5JQ(qWiU(ol^! z6Rc@G(lW*vb!fvX#HpiJE7NDWpoEY%jlKsWh>KQOQx{W;+b(_$sjwM7C#@i|xj_7U z@|wD~OId4h3V#t6?a*rI5ad?F9Xv6-t!@#_$}F1}#-QKU`jwE|!N%k5hMKPud2GVr zkqJw4nuWnMmvJW!)JjaNoE=u%72!d?ceroG$y62=vm%RIQBVejEXOS3$cX|8T{n&) zCCr6^;0OwWFeik@a$eDd*dj{WmsnUYs4A-RiyHbK*t)+2mMZ9Z;OqA#jlB`* zeYpm%YK7w8-Y@HeK9DENS~2o~|6oPK5x5zwY76!Kz{#)G+QI{G_OEM;QDt%C-de3u uEuSBs){1{9)^+Wx`t8T{+S~^}KGmgiTzhc``~?PvP4&`)f&!O<0s%3exJF_C From 1a1dba265b59b1163c07e10fbb5068eb6d14e1cf Mon Sep 17 00:00:00 2001 From: Christophe Antoniewski Date: Sat, 27 Jan 2024 16:09:16 +0100 Subject: [PATCH 07/16] specify ftype attribute to get lines_diff effective --- tools/htseq_count/htseq-count.xml | 2 +- 1 file changed, 1 insertion(+), 1 deletion(-) diff --git a/tools/htseq_count/htseq-count.xml b/tools/htseq_count/htseq-count.xml index 15034d4d930..95598363117 100644 --- a/tools/htseq_count/htseq-count.xml +++ b/tools/htseq_count/htseq-count.xml @@ -203,7 +203,7 @@ - + From 7fe5b00be8fe694c478dfee332f721da3d6aa577 Mon Sep 17 00:00:00 2001 From: Christophe Antoniewski Date: Sun, 28 Jan 2024 17:34:17 +0100 Subject: [PATCH 08/16] change variable othercounts to allcounts and fix versionning code --- tools/htseq_count/htseq-count.xml | 25 +++++++++++-------- ...ts.tsv => htseq-test-paired_allcounts.tsv} | 0 ...hercounts.tsv => htseq-test_allcounts.tsv} | 0 3 files changed, 15 insertions(+), 10 deletions(-) rename tools/htseq_count/test-data/{htseq-test-paired_othercounts.tsv => htseq-test-paired_allcounts.tsv} (100%) rename tools/htseq_count/test-data/{htseq-test_othercounts.tsv => htseq-test_allcounts.tsv} (100%) diff --git a/tools/htseq_count/htseq-count.xml b/tools/htseq_count/htseq-count.xml index 95598363117..42591a870f7 100644 --- a/tools/htseq_count/htseq-count.xml +++ b/tools/htseq_count/htseq-count.xml @@ -1,5 +1,10 @@ - + - Count aligned reads in a BAM file that overlap features in a GFF file + + 2.0.3 + 0 + 23.0 + htseq @@ -62,9 +67,9 @@ "$gfffile" | awk '{if ($1 ~ "no_feature|ambiguous|too_low_aQual|not_aligned|alignment_not_unique|aligned") print $0 | "cat 1>&2"; else print $0}' > '$counts' - 2> '$othercounts' && + 2> '$allcounts' && - echo -e "__aligned\t\$(cut -f2 '$counts' | awk '{s+=$1}END{print s}' -)" >> '$othercounts' && + echo -e "__aligned\t\$(cut -f2 '$counts' | awk '{s+=$1}END{print s}' -)" >> '$allcounts' && sed -i 's/\"//g' '$counts' #if str( $advanced_options.advanced_options_selector ) == "advanced": @@ -158,7 +163,7 @@ - + @@ -173,26 +178,26 @@ - + - + - + - + @@ -202,7 +207,7 @@ - + @@ -213,7 +218,7 @@ - + diff --git a/tools/htseq_count/test-data/htseq-test-paired_othercounts.tsv b/tools/htseq_count/test-data/htseq-test-paired_allcounts.tsv similarity index 100% rename from tools/htseq_count/test-data/htseq-test-paired_othercounts.tsv rename to tools/htseq_count/test-data/htseq-test-paired_allcounts.tsv diff --git a/tools/htseq_count/test-data/htseq-test_othercounts.tsv b/tools/htseq_count/test-data/htseq-test_allcounts.tsv similarity index 100% rename from tools/htseq_count/test-data/htseq-test_othercounts.tsv rename to tools/htseq_count/test-data/htseq-test_allcounts.tsv From ea46e211cf33a0b7f2cb4d9b003171245545175b Mon Sep 17 00:00:00 2001 From: Christophe Antoniewski Date: Sun, 28 Jan 2024 17:51:16 +0100 Subject: [PATCH 09/16] update README.rst and another URL in htseq-count.xml --- tools/htseq_count/README.rst | 2 +- tools/htseq_count/htseq-count.xml | 2 +- 2 files changed, 2 insertions(+), 2 deletions(-) diff --git a/tools/htseq_count/README.rst b/tools/htseq_count/README.rst index 732bb826095..30b5c22186f 100644 --- a/tools/htseq_count/README.rst +++ b/tools/htseq_count/README.rst @@ -1,7 +1,7 @@ htseq-count wrapper for Galaxy ============================== -Galaxy wrapper for `htseq-count `_ +Galaxy wrapper for `htseq-count `_ script from the `HTSEQ `_ python package. Installation diff --git a/tools/htseq_count/htseq-count.xml b/tools/htseq_count/htseq-count.xml index 42591a870f7..1e3f49bb020 100644 --- a/tools/htseq_count/htseq-count.xml +++ b/tools/htseq_count/htseq-count.xml @@ -315,7 +315,7 @@ Usage: htseq-count [options] sam_file gff_file This script takes an alignment file in SAM format and a feature file in GFF format and calculates for each feature the number of reads mapping to it. See -https://htseq.readthedocs.io/en/release_0.9.1/count.html for details. +https://htseq.readthedocs.io/en/latest/ for details. Options: -f , --format= From ff8c1e58b58f73decd540e7e1b7f2bb810b19912 Mon Sep 17 00:00:00 2001 From: Christophe Antoniewski Date: Mon, 29 Jan 2024 20:20:53 +0100 Subject: [PATCH 10/16] address changes suggested by @wm75 --- tools/htseq_count/htseq-count.xml | 22 +++++++++--------- .../test-data/htseq-test_samout.bam | Bin 66227 -> 66238 bytes 2 files changed, 11 insertions(+), 11 deletions(-) diff --git a/tools/htseq_count/htseq-count.xml b/tools/htseq_count/htseq-count.xml index 1e3f49bb020..e970960455b 100644 --- a/tools/htseq_count/htseq-count.xml +++ b/tools/htseq_count/htseq-count.xml @@ -1,7 +1,7 @@ - Count aligned reads in a BAM file that overlap features in a GFF file - 2.0.3 + 2.0.5 0 23.0 @@ -9,8 +9,9 @@ htseq - htseq + htseq samtools + gawk @@ -40,11 +41,13 @@ #end if #if $samfile.extension == 'bam': - samtools sort -n -T "\${TMPDIR:-.}" --output-fmt=SAM -o '$name_sorted_alignment_filename' '$samfile' && + samtools sort -n -T "\${TMPDIR:-.}" --output-fmt=BAM -o '$name_sorted_alignment_filename' '$samfile' && #else - samtools view -Su -t '${reference_fasta_filename}.fai' '$samfile' | samtools sort -n -T "\${TMPDIR:-.}" --output-fmt=SAM -o '$name_sorted_alignment_filename' - && + samtools view -Su -t '${reference_fasta_filename}.fai' '$samfile' | samtools sort -n -T "\${TMPDIR:-.}" --output-fmt=BAM -o '$name_sorted_alignment_filename' - && #end if + ln -s '$gfffile' reference.gtf && + htseq-count --mode=$mode --stranded=$stranded @@ -62,15 +65,12 @@ #end if --order=name - --format=sam '$name_sorted_alignment_filename' - "$gfffile" | awk '{if ($1 ~ "no_feature|ambiguous|too_low_aQual|not_aligned|alignment_not_unique|aligned") print $0 | "cat 1>&2"; else print $0}' - > '$counts' - 2> '$allcounts' && + reference.gtf | csplit -q - /^__/ && + mv xx00 '$counts' && mv xx01 '$allcounts' && - echo -e "__aligned\t\$(cut -f2 '$counts' | awk '{s+=$1}END{print s}' -)" >> '$allcounts' && - sed -i 's/\"//g' '$counts' + echo -e "__aligned\t\$(cut -f2 '$counts' 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