Does this pull request fix issue #24 ? by jelber2 · Pull Request #26 · OceanGenomics/mudskipper

jelber2 · 2022-05-17T08:23:35Z

Does this pull request fix issue #24 ?

get cDNA

wget ftp://ftp.ensemblgenomes.org/pub/metazoa/release-46/fasta/caenorhabditis_elegans/cdna/Caenorhabditis_elegans.WBcel235.cdna.all.fa.gz

use seqtk to isolate first cDNA sequence

seqtk seq -l0 Caenorhabditis_elegans.WBcel235.cdna.all.fa.gz |paste - - |head -n1 |tr '\t' '\n' > test.fa

rust setup

rustup show

Default host: x86_64-unknown-linux-gnu
rustup home:  /nfs/scistore16/itgrp/jelbers/.rustup

installed toolchains
--------------------

stable-x86_64-unknown-linux-gnu (default)
nightly-x86_64-unknown-linux-gnu

active toolchain
----------------

stable-x86_64-unknown-linux-gnu (default)
rustc 1.60.0 (7737e0b5c 2022-04-04)

mudskipper installation

cd ~/git/
git clone https://github.com/jelber2/mudskipper
cd mudskipper
git show

commit 993320bdc4528615311c2250d68c3d7a91c2efc4 (HEAD -> main, origin/main, origin/HEAD)

cargo build --release

get GTF file and genome

wget ftp://ftp.ensemblgenomes.org/pub/metazoa/release-46/gtf/caenorhabditis_elegans/Caenorhabditis_elegans.WBcel235.46.gtf.gz
gunzip Caenorhabditis_elegans.WBcel235.46.gtf.gz
wget ftp://ftp.ensemblgenomes.org/pub/metazoa/release-46/fasta/caenorhabditis_elegans/dna/Caenorhabditis_elegans.WBcel235.dna.toplevel.fa.gz
gunzip Caenorhabditis_elegans.WBcel235.dna.toplevel.fa.gz

STAR index genome

module load seqtk/20210125
# make sequences upper case (remove soft-masking)
seqtk seq -CU Caenorhabditis_elegans.WBcel235.dna.toplevel.fa > Caenorhabditis_elegans.WBcel235.dna.toplevel_uppercase.fa

module load STAR/2.7.9a
mkdir -p Caenorhabditis_elegans.WBcel235.dna.toplevel_Ensembl46_STAR-2.7.9a_sjdbOverhang18
STAR \
--genomeSAindexNbases 12 \
--limitGenomeGenerateRAM 100000000000 \
--runThreadN 34 \
--sjdbOverhang 18 \
--runMode genomeGenerate \
--genomeDir Caenorhabditis_elegans.WBcel235.dna.toplevel_Ensembl46_STAR-2.7.9a_sjdbOverhang18 \
--genomeFastaFiles Caenorhabditis_elegans.WBcel235.dna.toplevel_uppercase.fa \
--sjdbGTFfile Caenorhabditis_elegans.WBcel235.46.gtf

simulate one 150-bp read pair on the transcript Y110A7A.10.1 with BBTools/BBMap's randomreads.sh version 38.82 (any recent version should be fine too)

module load bbtools/38.82
randomreads.sh ref=test.fa out1=test_R1.fq.gz out2=test_R2.fq.gz \
reads=1 simplenames=t illuminanames=t paired=t addslash=t seed=1

test_R1.fq.gz

@0/1
ACAATATTTGATGTGTGGAACTTCTAAGGAAAATGCTGCGAATGCTCTGATTCATGCTCGCTATCGGATCGCTCAACTTGACAAGGCTGTTGGATTGAAAAGAGAGCCACAAGATGGTATTTTCTTGATTCGAGGATCCTCTTCTCATGG
+
6=5559=;9=A<>A?=@A?CD@A=D@B@=@?DDDCB?ABD<D>CBA?BDB;@?AABD>@B@:C?A>=9:???@:D?8B?C>@:>@C;@=<??=@AA>?7?ABA6?@A?E@?;>B>B;7<<=7A9C:><=:<@;9C;:<;<5555;67:65

test_R2.fq.gz

@0/2
GTTTTGATAGCATCCGCGTGGGTTTCAAGACTGAAGTGAGAGTAATAGCGAACGAAGTCACTTATAGTTGTCAGGTAGACATTGCTGTGCTCGAATCCCCATCCTTCTTCGTTTTGTTCGATTAGACAATGTGAGACACGTTCTCCGTGA
+
44788<4<<;9>>??B@AB@<<>6?>?=BAAC5=;<@><;5@?C?BA8B;>?>>>=;A<C@?;?>BAA??C>CA?B96@6:@@=>:@<B<>@AB@<:><<>C;=C6@?=@9@7?==6<?<:9@A:?>7:=?A;8=A995<444;;44446

STAR mapping

module load STAR/2.7.9a
STAR  --runThreadN 32 \
--genomeDir auxData/Caenorhabditis_elegans.WBcel235.dna.toplevel_Ensembl46_STAR-2.7.9a_sjdbOverhang18 \
--readFilesCommand zcat \
--outSAMtype BAM SortedByCoordinate \
--outFileNamePrefix mapped/ref/RNAseq.MUT1.rep4 \
--outTmpDir TMP/ref_RNAseq.MUT1.rep4/TEMP/ \
--outReadsUnmapped Fastx \
--readFilesIn test_R1.fastq.gz test_R2.fastq.gz

mudskipper index GTF

export PATH="/nfs/scistore16/itgrp/jelbers/git/mudskipper/target/release:$PATH"
mudskipper index --gtf auxData/Caenorhabditis_elegans.WBcel235.46.gtf --dir-index auxData/mudskipper

[2022-05-12T06:02:52Z INFO  mudskipper] Mudskipper started...
[2022-05-12T06:02:52Z INFO  mudskipper::annotation] reading the gtf file and building the tree.
[2022-05-12T06:02:59Z INFO  mudskipper::annotation] building the tree
[2022-05-12T06:02:59Z INFO  mudskipper::annotation] Time to build the tree: 7.234329464s
[2022-05-12T06:02:59Z INFO  mudskipper::annotation] saving the GTF index
[2022-05-12T06:03:03Z INFO  mudskipper::annotation] Done with saving the GTF index
[2022-05-12T06:03:03Z INFO  mudskipper] Mudskipper finished.

run mudskipper bulk

mudskipper bulk --threads 32 --alignment Aligned.sortedByCoord.out.bam --index auxData/mudskipper --out alignments.bam
[2022-05-17T08:21:36Z INFO  mudskipper] Mudskipper started...
[2022-05-17T08:21:36Z INFO  mudskipper::annotation] Loading parsed GTF...
[2022-05-17T08:21:36Z INFO  mudskipper::bam] Number of reference sequences: 61451
[2022-05-17T08:21:36Z INFO  mudskipper::bam] thread count: 32 in total
[2022-05-17T08:21:36Z INFO  mudskipper::bam] thread count: 16 for reading
[2022-05-17T08:21:36Z INFO  mudskipper::bam] thread count: 16 for writing
[2022-05-17T08:21:37Z INFO  mudskipper] Mudskipper finished.

Aligned.sortedByCoord.out.bam (no header)

0	99	I	5108012	255	5M47N145M	=	5108277	596	GTGAAATTTGCCGCCCAAGTGAAACTTCTCCGAATCGTTGTTAAAGATGGAAAATGCGGATTCAATACAGATTCATTAACTCACGATTCTGTTGTCAGAGTAATTGAATTTCATCGAAATATTTCTCTCAATATTTTCAATGATGCACTT	8;9=898:;@>B9DAD89AB;C?EDEH<?B8AE9AD>?ACBC8>:@98=CFB?A;A=?9>:D;@BAAE>>@8A<>@=<AE=G>:C@@8;;8B=>>D@E?88=;CA89::;?ABD>=@8:><8=C=E?@888?@A8D<8C@9=8?888888	NH:i:1	HI:i:1	AS:i:298	nM:i:2	NM:i:1	MD:Z:99T50	jM:B:c,21	jI:B:i,5108017,5108063	MC:Z:47M181N103M
0	147	I	5108277	255	47M181N103M	=	5108012	-596	GGGGCCACTTCAGCAGCGTATGATTCTCAGTGCAAAAAATGATCATGATTGGAGAGAACGCTTGGAAATGGAAAATTTGAGAGCAGTACATTTGGCATTTGAGAGAGGAGCCAAGCTATATGATTCAGCTCATCAAGAAATGGAACGAGC	33338333347333383><37;3;398:8:79<785786=6<333?=77???93A;5A=B:79>48>A83;;3678<>@7A=:::=34337=??=;9BA<3=3;>83:=;>;5<9>9=?=;@9;;A=@;@=<B5:878:=8848:93533	NH:i:1	HI:i:1	AS:i:298	nM:i:2	NM:i:1	MD:Z:5A144	jM:B:c,21	jI:B:i,5108324,5108504	MC:Z:5M47N145M

alignments.bam (no header)

0	99	Y110A7A.10.1	180	255	150M	=	398	368	GTGAAATTTGCCGCCCAAGTGAAACTTCTCCGAATCGTTGTTAAAGATGGAAAATGCGGATTCAATACAGATTCATTAACTCACGATTCTGTTGTCAGAGTAATTGAATTTCATCGAAATATTTCTCTCAATATTTTCAATGATGCACTT	8;9=898:;@>B9DAD89AB;C?EDEH<?B8AE9AD>?ACBC8>:@98=CFB?A;A=?9>:D;@BAAE>>@8A<>@=<AE=G>:C@@8;;8B=>>D@E?88=;CA89::;?ABD>=@8:><8=C=E?@888?@A8D<8C@9=8?888888	NH:i:1	HI:i:1	AS:i:298	nM:i:2	NM:i:1	MD:Z:99T50	jM:B:c,21	jI:B:i,5108017,5108063	MC:Z:47M181N103M
0	147	Y110A7A.10.1	398	255	150M	=	180	-368	GGGGCCACTTCAGCAGCGTATGATTCTCAGTGCAAAAAATGATCATGATTGGAGAGAACGCTTGGAAATGGAAAATTTGAGAGCAGTACATTTGGCATTTGAGAGAGGAGCCAAGCTATATGATTCAGCTCATCAAGAAATGGAACGAGC	33338333347333383><37;3;398:8:79<785786=6<333?=77???93A;5A=B:79>48>A83;;3678<>@7A=:::=34337=??=;9BA<3=3;>83:=;>;5<9>9=?=;@9;;A=@;@=<B5:878:=8848:93533	NH:i:1	HI:i:1	AS:i:298	nM:i:2	NM:i:1	MD:Z:5A144	jM:B:c,21	jI:B:i,5108324,5108504	MC:Z:5M47N145M
0	99	Y110A7A.10.2	170	255	150M	=	388	368	GTGAAATTTGCCGCCCAAGTGAAACTTCTCCGAATCGTTGTTAAAGATGGAAAATGCGGATTCAATACAGATTCATTAACTCACGATTCTGTTGTCAGAGTAATTGAATTTCATCGAAATATTTCTCTCAATATTTTCAATGATGCACTT	8;9=898:;@>B9DAD89AB;C?EDEH<?B8AE9AD>?ACBC8>:@98=CFB?A;A=?9>:D;@BAAE>>@8A<>@=<AE=G>:C@@8;;8B=>>D@E?88=;CA89::;?ABD>=@8:><8=C=E?@888?@A8D<8C@9=8?888888	NH:i:1	HI:i:1	AS:i:298	nM:i:2	NM:i:1	MD:Z:99T50	jM:B:c,21	jI:B:i,5108017,5108063	MC:Z:47M181N103M
0	147	Y110A7A.10.2	388	255	150M	=	170	-368	GGGGCCACTTCAGCAGCGTATGATTCTCAGTGCAAAAAATGATCATGATTGGAGAGAACGCTTGGAAATGGAAAATTTGAGAGCAGTACATTTGGCATTTGAGAGAGGAGCCAAGCTATATGATTCAGCTCATCAAGAAATGGAACGAGC	33338333347333383><37;3;398:8:79<785786=6<333?=77???93A;5A=B:79>48>A83;;3678<>@7A=:::=34337=??=;9BA<3=3;>83:=;>;5<9>9=?=;@9;;A=@;@=<B5:878:=8848:93533	NH:i:1	HI:i:1	AS:i:298	nM:i:2	NM:i:1	MD:Z:5A144	jM:B:c,21	jI:B:i,5108324,5108504	MC:Z:5M47N145M

jelber2 · 2022-05-17T08:28:29Z

On second thought, perhaps my quick fix may only work with when 100% reads are paired and 100% mates are mapped? I have not tested how it works when one mate is unmapped. EDIT if one mate is unmapped, the mapped mate is not transferred when using mudskipper bulk

rob-p · 2022-05-20T03:47:07Z

Thanks for this test case and PR @jelber2! @gmarcais, can we take a look at this?

janoppelt · 2022-06-14T22:30:40Z

On second thought, perhaps my quick fix may only work with when 100% reads are paired and 100% mates are mapped? I have not tested how it works when one mate is unmapped. EDIT if one mate is unmapped, the mapped mate is not transferred when using mudskipper bulk

I am pretty sure this is not just because of your fix but rather a combination with the rest of the mudskipper bulk code. Your solution works only if both mates are present in a name sorted SAM/BAM. If the SAM/BAM file is not name sorted mudskipper bulk treats all the reads as sort of pseudo-single-end reads - it duplicates each of the reads with the only difference being the TLEN sign (switches + for - and vice versa).

For example (modified from tests/NC_007114.7_12427.sam):

Paired, mapped, name sorted = works fine (output not copied)

J00153:160:HMTNMBBXX:6:1104:18893:12427	419	NC_007114.7	24289610	3	149M	=	24289724	264	GGCAGTGGTCTCTGTCCTGGATCATGTAGAGTGTCGTTGTCAACCTGCTCCTCGCCCCGCACATCGCCGAAAGTCATCCAGCCAGAAACTAGACGCACGTGATCGTTCAGAGAAAAGTCGACCCAAAGAGGATCTCCATCGCAGGGATG	AAFFFJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJFJJFJJJJJFJJJJJJJJJJJFFJJJFJJJJJJJJJFAFAFJJJJJJJJJA<A	NH:i:2	HI:i:2	AS:i:297	nM:i:0
J00153:160:HMTNMBBXX:6:1104:18893:12427	339	NC_007114.7	24289724	3	150M	=	24289610	-264	AAGTCGACCCAAAGAGGATCTCCATCGCAGGGATGAACTCAAACACAACCAGAGGCTAAACCTGGAGGACCTGCTCAGCCACAGCTGGCTGCCCCAAGACAGGTTCTCTGAATCACCAGACACTGTCCATTTTGGCCGCGATGCCTGGAC	FAFJJFAJFJJJJJJJJJJJJJJJFJJJJJJJJJJJJJJJJJJJFJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJFJFJJJJJJJJJJJJJFJJJJFJJJJJJJJJJJJJJJJJJJJJJJJJFFFAA	NH:i:2	HI:i:2	AS:i:297	nM:i:0
J00153:160:HMTNMBBXX:6:1104:18893:12428	419	NC_007114.7	24289610	3	149M	=	24289724	264	GGCAGTGGTCTCTGTCCTGGATCATGTAGAGTGTCGTTGTCAACCTGCTCCTCGCCCCGCACATCGCCGAAAGTCATCCAGCCAGAAACTAGACGCACGTGATCGTTCAGAGAAAAGTCGACCCAAAGAGGATCTCCATCGCAGGGATG	AAFFFJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJFJJFJJJJJFJJJJJJJJJJJFFJJJFJJJJJJJJJFAFAFJJJJJJJJJA<A	NH:i:2	HI:i:2	AS:i:297	nM:i:0
J00153:160:HMTNMBBXX:6:1104:18893:12428	339	NC_007114.7	24289724	3	150M	=	24289610	-264	AAGTCGACCCAAAGAGGATCTCCATCGCAGGGATGAACTCAAACACAACCAGAGGCTAAACCTGGAGGACCTGCTCAGCCACAGCTGGCTGCCCCAAGACAGGTTCTCTGAATCACCAGACACTGTCCATTTTGGCCGCGATGCCTGGAC	FAFJJFAJFJJJJJJJJJJJJJJJFJJJJJJJJJJJJJJJJJJJFJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJFJFJJJJJJJJJJJJJFJJJJFJJJJJJJJJJJJJJJJJJJJJJJJJFFFAA	NH:i:2	HI:i:2	AS:i:297	nM:i:0

Paired, mapped, not name sorted (but coordinate sorted) = doesn't work:

J00153:160:HMTNMBBXX:6:1104:18893:12427	419	NC_007114.7	24289610	3	149M	=	24289724	264	GGCAGTGGTCTCTGTCCTGGATCATGTAGAGTGTCGTTGTCAACCTGCTCCTCGCCCCGCACATCGCCGAAAGTCATCCAGCCAGAAACTAGACGCACGTGATCGTTCAGAGAAAAGTCGACCCAAAGAGGATCTCCATCGCAGGGATG	AAFFFJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJFJJFJJJJJFJJJJJJJJJJJFFJJJFJJJJJJJJJFAFAFJJJJJJJJJA<A	NH:i:2	HI:i:2	AS:i:297	nM:i:0
J00153:160:HMTNMBBXX:6:1104:18893:12428	419	NC_007114.7	24289610	3	149M	=	24289724	264	GGCAGTGGTCTCTGTCCTGGATCATGTAGAGTGTCGTTGTCAACCTGCTCCTCGCCCCGCACATCGCCGAAAGTCATCCAGCCAGAAACTAGACGCACGTGATCGTTCAGAGAAAAGTCGACCCAAAGAGGATCTCCATCGCAGGGATG	AAFFFJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJFJJFJJJJJFJJJJJJJJJJJFFJJJFJJJJJJJJJFAFAFJJJJJJJJJA<A	NH:i:2	HI:i:2	AS:i:297	nM:i:0
J00153:160:HMTNMBBXX:6:1104:18893:12427	339	NC_007114.7	24289724	3	150M	=	24289610	-264	AAGTCGACCCAAAGAGGATCTCCATCGCAGGGATGAACTCAAACACAACCAGAGGCTAAACCTGGAGGACCTGCTCAGCCACAGCTGGCTGCCCCAAGACAGGTTCTCTGAATCACCAGACACTGTCCATTTTGGCCGCGATGCCTGGAC	FAFJJFAJFJJJJJJJJJJJJJJJFJJJJJJJJJJJJJJJJJJJFJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJFJFJJJJJJJJJJJJJFJJJJFJJJJJJJJJJJJJJJJJJJJJJJJJFFFAA	NH:i:2	HI:i:2	AS:i:297	nM:i:0
J00153:160:HMTNMBBXX:6:1104:18893:12428	339	NC_007114.7	24289724	3	150M	=	24289610	-264	AAGTCGACCCAAAGAGGATCTCCATCGCAGGGATGAACTCAAACACAACCAGAGGCTAAACCTGGAGGACCTGCTCAGCCACAGCTGGCTGCCCCAAGACAGGTTCTCTGAATCACCAGACACTGTCCATTTTGGCCGCGATGCCTGGAC	FAFJJFAJFJJJJJJJJJJJJJJJFJJJJJJJJJJJJJJJJJJJFJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJFJFJJJJJJJJJJJJJFJJJJFJJJJJJJJJJJJJJJJJJJJJJJJJFFFAA	NH:i:2	HI:i:2	AS:i:297	nM:i:0

results in:

J00153:160:HMTNMBBXX:6:1104:18893:12427	419	XM_021473592.1	1294	3	149M	=	1294	149	GGCAGTGGTCTCTGTCCTGGATCATGTAGAGTGTCGTTGTCAACCTGCTCCTCGCCCCGCACATCGCCGAAAGTCATCCAGCCAGAAACTAGACGCACGTGATCGTTCAGAGAAAAGTCGACCCAAAGAGGATCTCCATCGCAGGGATG	AAFFFJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJFJJFJJJJJFJJJJJJJJJJJFFJJJFJJJJJJJJJFAFAFJJJJJJJJJA<A	NH:i:2	HI:i:2	AS:i:297	nM:i:0
J00153:160:HMTNMBBXX:6:1104:18893:12427	419	XM_021473592.1	1294	3	149M	=	1294	-149	GGCAGTGGTCTCTGTCCTGGATCATGTAGAGTGTCGTTGTCAACCTGCTCCTCGCCCCGCACATCGCCGAAAGTCATCCAGCCAGAAACTAGACGCACGTGATCGTTCAGAGAAAAGTCGACCCAAAGAGGATCTCCATCGCAGGGATG	AAFFFJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJFJJFJJJJJFJJJJJJJJJJJFFJJJFJJJJJJJJJFAFAFJJJJJJJJJA<A	NH:i:2	HI:i:2	AS:i:297	nM:i:0
J00153:160:HMTNMBBXX:6:1104:18893:12428	419	XM_021473592.1	1294	3	149M	=	1294	149	GGCAGTGGTCTCTGTCCTGGATCATGTAGAGTGTCGTTGTCAACCTGCTCCTCGCCCCGCACATCGCCGAAAGTCATCCAGCCAGAAACTAGACGCACGTGATCGTTCAGAGAAAAGTCGACCCAAAGAGGATCTCCATCGCAGGGATG	AAFFFJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJFJJFJJJJJFJJJJJJJJJJJFFJJJFJJJJJJJJJFAFAFJJJJJJJJJA<A	NH:i:2	HI:i:2	AS:i:297	nM:i:0
J00153:160:HMTNMBBXX:6:1104:18893:12428	419	XM_021473592.1	1294	3	149M	=	1294	-149	GGCAGTGGTCTCTGTCCTGGATCATGTAGAGTGTCGTTGTCAACCTGCTCCTCGCCCCGCACATCGCCGAAAGTCATCCAGCCAGAAACTAGACGCACGTGATCGTTCAGAGAAAAGTCGACCCAAAGAGGATCTCCATCGCAGGGATG	AAFFFJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJFJJFJJJJJFJJJJJJJJJJJFFJJJFJJJJJJJJJFAFAFJJJJJJJJJA<A	NH:i:2	HI:i:2	AS:i:297	nM:i:0
J00153:160:HMTNMBBXX:6:1104:18893:12427	339	XM_021473592.1	1408	3	150M	=	1408	150	AAGTCGACCCAAAGAGGATCTCCATCGCAGGGATGAACTCAAACACAACCAGAGGCTAAACCTGGAGGACCTGCTCAGCCACAGCTGGCTGCCCCAAGACAGGTTCTCTGAATCACCAGACACTGTCCATTTTGGCCGCGATGCCTGGAC	FAFJJFAJFJJJJJJJJJJJJJJJFJJJJJJJJJJJJJJJJJJJFJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJFJFJJJJJJJJJJJJJFJJJJFJJJJJJJJJJJJJJJJJJJJJJJJJFFFAA	NH:i:2	HI:i:2	AS:i:297	nM:i:0
J00153:160:HMTNMBBXX:6:1104:18893:12427	339	XM_021473592.1	1408	3	150M	=	1408	-150	AAGTCGACCCAAAGAGGATCTCCATCGCAGGGATGAACTCAAACACAACCAGAGGCTAAACCTGGAGGACCTGCTCAGCCACAGCTGGCTGCCCCAAGACAGGTTCTCTGAATCACCAGACACTGTCCATTTTGGCCGCGATGCCTGGAC	FAFJJFAJFJJJJJJJJJJJJJJJFJJJJJJJJJJJJJJJJJJJFJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJFJFJJJJJJJJJJJJJFJJJJFJJJJJJJJJJJJJJJJJJJJJJJJJFFFAA	NH:i:2	HI:i:2	AS:i:297	nM:i:0
J00153:160:HMTNMBBXX:6:1104:18893:12428	339	XM_021473592.1	1408	3	150M	=	1408	150	AAGTCGACCCAAAGAGGATCTCCATCGCAGGGATGAACTCAAACACAACCAGAGGCTAAACCTGGAGGACCTGCTCAGCCACAGCTGGCTGCCCCAAGACAGGTTCTCTGAATCACCAGACACTGTCCATTTTGGCCGCGATGCCTGGAC	FAFJJFAJFJJJJJJJJJJJJJJJFJJJJJJJJJJJJJJJJJJJFJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJFJFJJJJJJJJJJJJJFJJJJFJJJJJJJJJJJJJJJJJJJJJJJJJFFFAA	NH:i:2	HI:i:2	AS:i:297	nM:i:0
J00153:160:HMTNMBBXX:6:1104:18893:12428	339	XM_021473592.1	1408	3	150M	=	1408	-150	AAGTCGACCCAAAGAGGATCTCCATCGCAGGGATGAACTCAAACACAACCAGAGGCTAAACCTGGAGGACCTGCTCAGCCACAGCTGGCTGCCCCAAGACAGGTTCTCTGAATCACCAGACACTGTCCATTTTGGCCGCGATGCCTGGAC	FAFJJFAJFJJJJJJJJJJJJJJJFJJJJJJJJJJJJJJJJJJJFJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJFJFJJJJJJJJJJJJJFJJJJFJJJJJJJJJJJJJJJJJJJJJJJJJFFFAA	NH:i:2	HI:i:2	AS:i:297	nM:i:0

One pair is mapped and both mates are present, the other pair is missing a mate completely (due to post-alignment filtering) but SAM/BAM is name sorted = the complete pair is correctly converted, the one-mate-pair read gets copied again with only the TLEN sign changed:

J00153:160:HMTNMBBXX:6:1104:18893:12428	419	NC_007114.7	24289610	3	149M	=	24289724	264	GGCAGTGGTCTCTGTCCTGGATCATGTAGAGTGTCGTTGTCAACCTGCTCCTCGCCCCGCACATCGCCGAAAGTCATCCAGCCAGAAACTAGACGCACGTGATCGTTCAGAGAAAAGTCGACCCAAAGAGGATCTCCATCGCAGGGATG	AAFFFJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJFJJFJJJJJFJJJJJJJJJJJFFJJJFJJJJJJJJJFAFAFJJJJJJJJJA<A	NH:i:2	HI:i:2	AS:i:297	nM:i:0
J00153:160:HMTNMBBXX:6:1104:18893:12428	339	NC_007114.7	24289724	3	150M	=	24289610	-264	AAGTCGACCCAAAGAGGATCTCCATCGCAGGGATGAACTCAAACACAACCAGAGGCTAAACCTGGAGGACCTGCTCAGCCACAGCTGGCTGCCCCAAGACAGGTTCTCTGAATCACCAGACACTGTCCATTTTGGCCGCGATGCCTGGAC	FAFJJFAJFJJJJJJJJJJJJJJJFJJJJJJJJJJJJJJJJJJJFJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJFJFJJJJJJJJJJJJJFJJJJFJJJJJJJJJJJJJJJJJJJJJJJJJFFFAA	NH:i:2	HI:i:2	AS:i:297	nM:i:0
J00153:160:HMTNMBBXX:6:1104:18893:12427	339	NC_007114.7	24289724	3	150M	=	24289610	-264	AAGTCGACCCAAAGAGGATCTCCATCGCAGGGATGAACTCAAACACAACCAGAGGCTAAACCTGGAGGACCTGCTCAGCCACAGCTGGCTGCCCCAAGACAGGTTCTCTGAATCACCAGACACTGTCCATTTTGGCCGCGATGCCTGGAC	FAFJJFAJFJJJJJJJJJJJJJJJFJJJJJJJJJJJJJJJJJJJFJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJFJFJJJJJJJJJJJJJFJJJJFJJJJJJJJJJJJJJJJJJJJJJJJJFFFAA	NH:i:2	HI:i:2	AS:i:297	nM:i:0

results in:

J00153:160:HMTNMBBXX:6:1104:18893:12428	419	XM_021473592.1	1294	3	149M	=	1408	264	GGCAGTGGTCTCTGTCCTGGATCATGTAGAGTGTCGTTGTCAACCTGCTCCTCGCCCCGCACATCGCCGAAAGTCATCCAGCCAGAAACTAGACGCACGTGATCGTTCAGAGAAAAGTCGACCCAAAGAGGATCTCCATCGCAGGGATG	AAFFFJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJFJJFJJJJJFJJJJJJJJJJJFFJJJFJJJJJJJJJFAFAFJJJJJJJJJA<A	NH:i:2	HI:i:2	AS:i:297	nM:i:0
J00153:160:HMTNMBBXX:6:1104:18893:12428	339	XM_021473592.1	1408	3	150M	=	1294	-264	AAGTCGACCCAAAGAGGATCTCCATCGCAGGGATGAACTCAAACACAACCAGAGGCTAAACCTGGAGGACCTGCTCAGCCACAGCTGGCTGCCCCAAGACAGGTTCTCTGAATCACCAGACACTGTCCATTTTGGCCGCGATGCCTGGAC	FAFJJFAJFJJJJJJJJJJJJJJJFJJJJJJJJJJJJJJJJJJJFJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJFJFJJJJJJJJJJJJJFJJJJFJJJJJJJJJJJJJJJJJJJJJJJJJFFFAA	NH:i:2	HI:i:2	AS:i:297	nM:i:0
J00153:160:HMTNMBBXX:6:1104:18893:12427	339	XM_021473592.1	1408	3	150M	=	1408	150	AAGTCGACCCAAAGAGGATCTCCATCGCAGGGATGAACTCAAACACAACCAGAGGCTAAACCTGGAGGACCTGCTCAGCCACAGCTGGCTGCCCCAAGACAGGTTCTCTGAATCACCAGACACTGTCCATTTTGGCCGCGATGCCTGGAC	FAFJJFAJFJJJJJJJJJJJJJJJFJJJJJJJJJJJJJJJJJJJFJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJFJFJJJJJJJJJJJJJFJJJJFJJJJJJJJJJJJJJJJJJJJJJJJJFFFAA	NH:i:2	HI:i:2	AS:i:297	nM:i:0
J00153:160:HMTNMBBXX:6:1104:18893:12427	339	XM_021473592.1	1408	3	150M	=	1408	-150	AAGTCGACCCAAAGAGGATCTCCATCGCAGGGATGAACTCAAACACAACCAGAGGCTAAACCTGGAGGACCTGCTCAGCCACAGCTGGCTGCCCCAAGACAGGTTCTCTGAATCACCAGACACTGTCCATTTTGGCCGCGATGCCTGGAC	FAFJJFAJFJJJJJJJJJJJJJJJFJJJJJJJJJJJJJJJJJJJFJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJFJFJJJJJJJJJJJJJFJJJJFJJJJJJJJJJJJJJJJJJJJJJJJJFFFAA	NH:i:2	HI:i:2	AS:i:297	nM:i:0

jelber2 · 2022-06-17T12:04:30Z

Dear Jan, thanks for catching this. I can see these trends in my 'real' data too. Using this pull request as is.

jelber2 · 2022-06-20T08:47:14Z

@opplatek, please try your tests again, as I think I made some progress! It may even work now whether the BAM is coordinate or name sorted

jelber2 · 2022-06-20T09:21:09Z

Note that I added your test examples in tests/

Paired, mapped, name sorted
target/release/mudskipper bulk --alignment tests/NC_007114.7_12427_1.sam --gtf tests/NC_007114.7_12427.gtf --out test1.bam

cat <(grep -v "^@" tests/NC_007114.7_12427_1.sam) <(echo) <(samtools view test1.bam)

J00153:160:HMTNMBBXX:6:1104:18893:12427	419	NC_007114.7	24289610	3	149M	=	24289724	264	GGCAGTGGTCTCTGTCCTGGATCATGTAGAGTGTCGTTGTCAACCTGCTCCTCGCCCCGCACATCGCCGAAAGTCATCCAGCCAGAAACTAGACGCACGTGATCGTTCAGAGAAAAGTCGACCCAAAGAGGATCTCCATCGCAGGGATG	AAFFFJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJFJJFJJJJJFJJJJJJJJJJJFFJJJFJJJJJJJJJFAFAFJJJJJJJJJA<A	NH:i:2	HI:i:2	AS:i:297	nM:i:0
J00153:160:HMTNMBBXX:6:1104:18893:12427	339	NC_007114.7	24289724	3	150M	=	24289610	-264	AAGTCGACCCAAAGAGGATCTCCATCGCAGGGATGAACTCAAACACAACCAGAGGCTAAACCTGGAGGACCTGCTCAGCCACAGCTGGCTGCCCCAAGACAGGTTCTCTGAATCACCAGACACTGTCCATTTTGGCCGCGATGCCTGGAC	FAFJJFAJFJJJJJJJJJJJJJJJFJJJJJJJJJJJJJJJJJJJFJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJFJFJJJJJJJJJJJJJFJJJJFJJJJJJJJJJJJJJJJJJJJJJJJJFFFAA	NH:i:2	HI:i:2	AS:i:297	nM:i:0
J00153:160:HMTNMBBXX:6:1104:18893:12428	419	NC_007114.7	24289610	3	149M	=	24289724	264	GGCAGTGGTCTCTGTCCTGGATCATGTAGAGTGTCGTTGTCAACCTGCTCCTCGCCCCGCACATCGCCGAAAGTCATCCAGCCAGAAACTAGACGCACGTGATCGTTCAGAGAAAAGTCGACCCAAAGAGGATCTCCATCGCAGGGATG	AAFFFJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJFJJFJJJJJFJJJJJJJJJJJFFJJJFJJJJJJJJJFAFAFJJJJJJJJJA<A	NH:i:2	HI:i:2	AS:i:297	nM:i:0
J00153:160:HMTNMBBXX:6:1104:18893:12428	339	NC_007114.7	24289724	3	150M	=	24289610	-264	AAGTCGACCCAAAGAGGATCTCCATCGCAGGGATGAACTCAAACACAACCAGAGGCTAAACCTGGAGGACCTGCTCAGCCACAGCTGGCTGCCCCAAGACAGGTTCTCTGAATCACCAGACACTGTCCATTTTGGCCGCGATGCCTGGAC	FAFJJFAJFJJJJJJJJJJJJJJJFJJJJJJJJJJJJJJJJJJJFJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJFJFJJJJJJJJJJJJJFJJJJFJJJJJJJJJJJJJJJJJJJJJJJJJFFFAA	NH:i:2	HI:i:2	AS:i:297	nM:i:0

J00153:160:HMTNMBBXX:6:1104:18893:12427	419	XM_021473592.1	1294	3	149M	=	1408	264	GGCAGTGGTCTCTGTCCTGGATCATGTAGAGTGTCGTTGTCAACCTGCTCCTCGCCCCGCACATCGCCGAAAGTCATCCAGCCAGAAACTAGACGCACGTGATCGTTCAGAGAAAAGTCGACCCAAAGAGGATCTCCATCGCAGGGATG	AAFFFJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJFJJFJJJJJFJJJJJJJJJJJFFJJJFJJJJJJJJJFAFAFJJJJJJJJJA<A	NH:i:2	HI:i:2	AS:i:297	nM:i:0
J00153:160:HMTNMBBXX:6:1104:18893:12427	339	XM_021473592.1	1408	3	150M	=	1294	-264	AAGTCGACCCAAAGAGGATCTCCATCGCAGGGATGAACTCAAACACAACCAGAGGCTAAACCTGGAGGACCTGCTCAGCCACAGCTGGCTGCCCCAAGACAGGTTCTCTGAATCACCAGACACTGTCCATTTTGGCCGCGATGCCTGGAC	FAFJJFAJFJJJJJJJJJJJJJJJFJJJJJJJJJJJJJJJJJJJFJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJFJFJJJJJJJJJJJJJFJJJJFJJJJJJJJJJJJJJJJJJJJJJJJJFFFAA	NH:i:2	HI:i:2	AS:i:297	nM:i:0
J00153:160:HMTNMBBXX:6:1104:18893:12428	419	XM_021473592.1	1294	3	149M	=	1408	264	GGCAGTGGTCTCTGTCCTGGATCATGTAGAGTGTCGTTGTCAACCTGCTCCTCGCCCCGCACATCGCCGAAAGTCATCCAGCCAGAAACTAGACGCACGTGATCGTTCAGAGAAAAGTCGACCCAAAGAGGATCTCCATCGCAGGGATG	AAFFFJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJFJJFJJJJJFJJJJJJJJJJJFFJJJFJJJJJJJJJFAFAFJJJJJJJJJA<A	NH:i:2	HI:i:2	AS:i:297	nM:i:0
J00153:160:HMTNMBBXX:6:1104:18893:12428	339	XM_021473592.1	1408	3	150M	=	1294	-264	AAGTCGACCCAAAGAGGATCTCCATCGCAGGGATGAACTCAAACACAACCAGAGGCTAAACCTGGAGGACCTGCTCAGCCACAGCTGGCTGCCCCAAGACAGGTTCTCTGAATCACCAGACACTGTCCATTTTGGCCGCGATGCCTGGAC	FAFJJFAJFJJJJJJJJJJJJJJJFJJJJJJJJJJJJJJJJJJJFJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJFJFJJJJJJJJJJJJJFJJJJFJJJJJJJJJJJJJJJJJJJJJJJJJFFFAA	NH:i:2	HI:i:2	AS:i:297	nM:i:0

Paired, mapped, coordinate-sorted has incorrect position of next read in mudskipper bam
target/release/mudskipper bulk --alignment tests/NC_007114.7_12427_2.sam --gtf tests/NC_007114.7_12427.gtf --out test2.bam

cat <(grep -v "^@" tests/NC_007114.7_12427_2.sam) <(echo) <(samtools view test2.bam)

J00153:160:HMTNMBBXX:6:1104:18893:12427	419	NC_007114.7	24289610	3	149M	=	24289724	264	GGCAGTGGTCTCTGTCCTGGATCATGTAGAGTGTCGTTGTCAACCTGCTCCTCGCCCCGCACATCGCCGAAAGTCATCCAGCCAGAAACTAGACGCACGTGATCGTTCAGAGAAAAGTCGACCCAAAGAGGATCTCCATCGCAGGGATG	AAFFFJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJFJJFJJJJJFJJJJJJJJJJJFFJJJFJJJJJJJJJFAFAFJJJJJJJJJA<A	NH:i:2	HI:i:2	AS:i:297	nM:i:0
J00153:160:HMTNMBBXX:6:1104:18893:12428	419	NC_007114.7	24289610	3	149M	=	24289724	264	GGCAGTGGTCTCTGTCCTGGATCATGTAGAGTGTCGTTGTCAACCTGCTCCTCGCCCCGCACATCGCCGAAAGTCATCCAGCCAGAAACTAGACGCACGTGATCGTTCAGAGAAAAGTCGACCCAAAGAGGATCTCCATCGCAGGGATG	AAFFFJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJFJJFJJJJJFJJJJJJJJJJJFFJJJFJJJJJJJJJFAFAFJJJJJJJJJA<A	NH:i:2	HI:i:2	AS:i:297	nM:i:0
J00153:160:HMTNMBBXX:6:1104:18893:12427	339	NC_007114.7	24289724	3	150M	=	24289610	-264	AAGTCGACCCAAAGAGGATCTCCATCGCAGGGATGAACTCAAACACAACCAGAGGCTAAACCTGGAGGACCTGCTCAGCCACAGCTGGCTGCCCCAAGACAGGTTCTCTGAATCACCAGACACTGTCCATTTTGGCCGCGATGCCTGGAC	FAFJJFAJFJJJJJJJJJJJJJJJFJJJJJJJJJJJJJJJJJJJFJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJFJFJJJJJJJJJJJJJFJJJJFJJJJJJJJJJJJJJJJJJJJJJJJJFFFAA	NH:i:2	HI:i:2	AS:i:297	nM:i:0
J00153:160:HMTNMBBXX:6:1104:18893:12428	339	NC_007114.7	24289724	3	150M	=	24289610	-264	AAGTCGACCCAAAGAGGATCTCCATCGCAGGGATGAACTCAAACACAACCAGAGGCTAAACCTGGAGGACCTGCTCAGCCACAGCTGGCTGCCCCAAGACAGGTTCTCTGAATCACCAGACACTGTCCATTTTGGCCGCGATGCCTGGAC	FAFJJFAJFJJJJJJJJJJJJJJJFJJJJJJJJJJJJJJJJJJJFJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJFJFJJJJJJJJJJJJJFJJJJFJJJJJJJJJJJJJJJJJJJJJJJJJFFFAA	NH:i:2	HI:i:2	AS:i:297	nM:i:0

J00153:160:HMTNMBBXX:6:1104:18893:12427	419	XM_021473592.1	1294	3	149M	=	24289724	264	GGCAGTGGTCTCTGTCCTGGATCATGTAGAGTGTCGTTGTCAACCTGCTCCTCGCCCCGCACATCGCCGAAAGTCATCCAGCCAGAAACTAGACGCACGTGATCGTTCAGAGAAAAGTCGACCCAAAGAGGATCTCCATCGCAGGGATG	AAFFFJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJFJJFJJJJJFJJJJJJJJJJJFFJJJFJJJJJJJJJFAFAFJJJJJJJJJA<A	NH:i:2	HI:i:2	AS:i:297	nM:i:0
J00153:160:HMTNMBBXX:6:1104:18893:12428	419	XM_021473592.1	1294	3	149M	=	24289724	264	GGCAGTGGTCTCTGTCCTGGATCATGTAGAGTGTCGTTGTCAACCTGCTCCTCGCCCCGCACATCGCCGAAAGTCATCCAGCCAGAAACTAGACGCACGTGATCGTTCAGAGAAAAGTCGACCCAAAGAGGATCTCCATCGCAGGGATG	AAFFFJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJFJJFJJJJJFJJJJJJJJJJJFFJJJFJJJJJJJJJFAFAFJJJJJJJJJA<A	NH:i:2	HI:i:2	AS:i:297	nM:i:0
J00153:160:HMTNMBBXX:6:1104:18893:12427	339	XM_021473592.1	1408	3	150M	=	24289610	-264	AAGTCGACCCAAAGAGGATCTCCATCGCAGGGATGAACTCAAACACAACCAGAGGCTAAACCTGGAGGACCTGCTCAGCCACAGCTGGCTGCCCCAAGACAGGTTCTCTGAATCACCAGACACTGTCCATTTTGGCCGCGATGCCTGGAC	FAFJJFAJFJJJJJJJJJJJJJJJFJJJJJJJJJJJJJJJJJJJFJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJFJFJJJJJJJJJJJJJFJJJJFJJJJJJJJJJJJJJJJJJJJJJJJJFFFAA	NH:i:2	HI:i:2	AS:i:297	nM:i:0
J00153:160:HMTNMBBXX:6:1104:18893:12428	339	XM_021473592.1	1408	3	150M	=	24289610	-264	AAGTCGACCCAAAGAGGATCTCCATCGCAGGGATGAACTCAAACACAACCAGAGGCTAAACCTGGAGGACCTGCTCAGCCACAGCTGGCTGCCCCAAGACAGGTTCTCTGAATCACCAGACACTGTCCATTTTGGCCGCGATGCCTGGAC	FAFJJFAJFJJJJJJJJJJJJJJJFJJJJJJJJJJJJJJJJJJJFJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJFJFJJJJJJJJJJJJJFJJJJFJJJJJJJJJJJJJJJJJJJJJJJJJFFFAA	NH:i:2	HI:i:2	AS:i:297	nM:i:0

One pair is mapped and both mates are present, the other pair is missing a mate completely (due to post-alignment filtering) but SAM/BAM is name sorted = the complete pair is correctly converted, the one-mate-pair read has incorrect position of next read in mudskipper bam
target/release/mudskipper bulk --alignment tests/NC_007114.7_12427_3.sam --gtf tests/NC_007114.7_12427.gtf --out test3.bam

cat <(grep -v "^@" tests/NC_007114.7_12427_3.sam) <(echo) <(samtools view test3.bam)

J00153:160:HMTNMBBXX:6:1104:18893:12428	419	NC_007114.7	24289610	3	149M	=	24289724	264	GGCAGTGGTCTCTGTCCTGGATCATGTAGAGTGTCGTTGTCAACCTGCTCCTCGCCCCGCACATCGCCGAAAGTCATCCAGCCAGAAACTAGACGCACGTGATCGTTCAGAGAAAAGTCGACCCAAAGAGGATCTCCATCGCAGGGATG	AAFFFJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJFJJFJJJJJFJJJJJJJJJJJFFJJJFJJJJJJJJJFAFAFJJJJJJJJJA<A	NH:i:2	HI:i:2	AS:i:297	nM:i:0
J00153:160:HMTNMBBXX:6:1104:18893:12428	339	NC_007114.7	24289724	3	150M	=	24289610	-264	AAGTCGACCCAAAGAGGATCTCCATCGCAGGGATGAACTCAAACACAACCAGAGGCTAAACCTGGAGGACCTGCTCAGCCACAGCTGGCTGCCCCAAGACAGGTTCTCTGAATCACCAGACACTGTCCATTTTGGCCGCGATGCCTGGAC	FAFJJFAJFJJJJJJJJJJJJJJJFJJJJJJJJJJJJJJJJJJJFJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJFJFJJJJJJJJJJJJJFJJJJFJJJJJJJJJJJJJJJJJJJJJJJJJFFFAA	NH:i:2	HI:i:2	AS:i:297	nM:i:0
J00153:160:HMTNMBBXX:6:1104:18893:12427	339	NC_007114.7	24289724	3	150M	=	24289610	-264	AAGTCGACCCAAAGAGGATCTCCATCGCAGGGATGAACTCAAACACAACCAGAGGCTAAACCTGGAGGACCTGCTCAGCCACAGCTGGCTGCCCCAAGACAGGTTCTCTGAATCACCAGACACTGTCCATTTTGGCCGCGATGCCTGGAC	FAFJJFAJFJJJJJJJJJJJJJJJFJJJJJJJJJJJJJJJJJJJFJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJFJFJJJJJJJJJJJJJFJJJJFJJJJJJJJJJJJJJJJJJJJJJJJJFFFAA	NH:i:2	HI:i:2	AS:i:297	nM:i:0

J00153:160:HMTNMBBXX:6:1104:18893:12428	419	XM_021473592.1	1294	3	149M	=	1408	264	GGCAGTGGTCTCTGTCCTGGATCATGTAGAGTGTCGTTGTCAACCTGCTCCTCGCCCCGCACATCGCCGAAAGTCATCCAGCCAGAAACTAGACGCACGTGATCGTTCAGAGAAAAGTCGACCCAAAGAGGATCTCCATCGCAGGGATG	AAFFFJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJFJJFJJJJJFJJJJJJJJJJJFFJJJFJJJJJJJJJFAFAFJJJJJJJJJA<A	NH:i:2	HI:i:2	AS:i:297	nM:i:0
J00153:160:HMTNMBBXX:6:1104:18893:12428	339	XM_021473592.1	1408	3	150M	=	1294	-264	AAGTCGACCCAAAGAGGATCTCCATCGCAGGGATGAACTCAAACACAACCAGAGGCTAAACCTGGAGGACCTGCTCAGCCACAGCTGGCTGCCCCAAGACAGGTTCTCTGAATCACCAGACACTGTCCATTTTGGCCGCGATGCCTGGAC	FAFJJFAJFJJJJJJJJJJJJJJJFJJJJJJJJJJJJJJJJJJJFJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJFJFJJJJJJJJJJJJJFJJJJFJJJJJJJJJJJJJJJJJJJJJJJJJFFFAA	NH:i:2	HI:i:2	AS:i:297	nM:i:0
J00153:160:HMTNMBBXX:6:1104:18893:12427	339	XM_021473592.1	1408	3	150M	=	24289610	-264	AAGTCGACCCAAAGAGGATCTCCATCGCAGGGATGAACTCAAACACAACCAGAGGCTAAACCTGGAGGACCTGCTCAGCCACAGCTGGCTGCCCCAAGACAGGTTCTCTGAATCACCAGACACTGTCCATTTTGGCCGCGATGCCTGGAC	FAFJJFAJFJJJJJJJJJJJJJJJFJJJJJJJJJJJJJJJJJJJFJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJFJFJJJJJJJJJJJJJFJJJJFJJJJJJJJJJJJJJJJJJJJJJJJJFFFAA	NH:i:2	HI:i:2	AS:i:297	nM:i:0

jelber2 · 2022-06-20T10:53:40Z

Well, it will take me a while to figure out why the singletons have incorrect position of next read in mudskipper bam values. Sorting by query name before mudskipper only works if both mates are present.

jelber2 · 2022-06-21T07:26:08Z

Ok, well, I think I fixed it for now, but one must still do sorting by read name prior to running mudskipper.

Paired, mapped, coordinate-sorted must sort by read name, otherwise, pairs not next to each other are considered single-end reads
target/release/mudskipper bulk --alignment tests/NC_007114.7_12427_2.sam --gtf tests/NC_007114.7_12427.gtf --out test2.bam

cat <(grep -v "^@" tests/NC_007114.7_12427_2.sam) <(echo) <(samtools view test2.bam)

J00153:160:HMTNMBBXX:6:1104:18893:12427	419	NC_007114.7	24289610	3	149M	=	24289724	264	GGCAGTGGTCTCTGTCCTGGATCATGTAGAGTGTCGTTGTCAACCTGCTCCTCGCCCCGCACATCGCCGAAAGTCATCCAGCCAGAAACTAGACGCACGTGATCGTTCAGAGAAAAGTCGACCCAAAGAGGATCTCCATCGCAGGGATG	AAFFFJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJFJJFJJJJJFJJJJJJJJJJJFFJJJFJJJJJJJJJFAFAFJJJJJJJJJA<A	NH:i:2	HI:i:2	AS:i:297	nM:i:0
J00153:160:HMTNMBBXX:6:1104:18893:12428	419	NC_007114.7	24289610	3	149M	=	24289724	264	GGCAGTGGTCTCTGTCCTGGATCATGTAGAGTGTCGTTGTCAACCTGCTCCTCGCCCCGCACATCGCCGAAAGTCATCCAGCCAGAAACTAGACGCACGTGATCGTTCAGAGAAAAGTCGACCCAAAGAGGATCTCCATCGCAGGGATG	AAFFFJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJFJJFJJJJJFJJJJJJJJJJJFFJJJFJJJJJJJJJFAFAFJJJJJJJJJA<A	NH:i:2	HI:i:2	AS:i:297	nM:i:0
J00153:160:HMTNMBBXX:6:1104:18893:12427	339	NC_007114.7	24289724	3	150M	=	24289610	-264	AAGTCGACCCAAAGAGGATCTCCATCGCAGGGATGAACTCAAACACAACCAGAGGCTAAACCTGGAGGACCTGCTCAGCCACAGCTGGCTGCCCCAAGACAGGTTCTCTGAATCACCAGACACTGTCCATTTTGGCCGCGATGCCTGGAC	FAFJJFAJFJJJJJJJJJJJJJJJFJJJJJJJJJJJJJJJJJJJFJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJFJFJJJJJJJJJJJJJFJJJJFJJJJJJJJJJJJJJJJJJJJJJJJJFFFAA	NH:i:2	HI:i:2	AS:i:297	nM:i:0
J00153:160:HMTNMBBXX:6:1104:18893:12428	339	NC_007114.7	24289724	3	150M	=	24289610	-264	AAGTCGACCCAAAGAGGATCTCCATCGCAGGGATGAACTCAAACACAACCAGAGGCTAAACCTGGAGGACCTGCTCAGCCACAGCTGGCTGCCCCAAGACAGGTTCTCTGAATCACCAGACACTGTCCATTTTGGCCGCGATGCCTGGAC	FAFJJFAJFJJJJJJJJJJJJJJJFJJJJJJJJJJJJJJJJJJJFJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJFJFJJJJJJJJJJJJJFJJJJFJJJJJJJJJJJJJJJJJJJJJJJJJFFFAA	NH:i:2	HI:i:2	AS:i:297	nM:i:0

J00153:160:HMTNMBBXX:6:1104:18893:12427	418	XM_021473592.1	1294	3	149M	*	0	0	GGCAGTGGTCTCTGTCCTGGATCATGTAGAGTGTCGTTGTCAACCTGCTCCTCGCCCCGCACATCGCCGAAAGTCATCCAGCCAGAAACTAGACGCACGTGATCGTTCAGAGAAAAGTCGACCCAAAGAGGATCTCCATCGCAGGGATG	AAFFFJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJFJJFJJJJJFJJJJJJJJJJJFFJJJFJJJJJJJJJFAFAFJJJJJJJJJA<A	NH:i:2	HI:i:2	AS:i:297	nM:i:0
J00153:160:HMTNMBBXX:6:1104:18893:12428	418	XM_021473592.1	1294	3	149M	*	0	0	GGCAGTGGTCTCTGTCCTGGATCATGTAGAGTGTCGTTGTCAACCTGCTCCTCGCCCCGCACATCGCCGAAAGTCATCCAGCCAGAAACTAGACGCACGTGATCGTTCAGAGAAAAGTCGACCCAAAGAGGATCTCCATCGCAGGGATG	AAFFFJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJFJJFJJJJJFJJJJJJJJJJJFFJJJFJJJJJJJJJFAFAFJJJJJJJJJA<A	NH:i:2	HI:i:2	AS:i:297	nM:i:0
J00153:160:HMTNMBBXX:6:1104:18893:12427	338	XM_021473592.1	1408	3	150M	*	0	0	AAGTCGACCCAAAGAGGATCTCCATCGCAGGGATGAACTCAAACACAACCAGAGGCTAAACCTGGAGGACCTGCTCAGCCACAGCTGGCTGCCCCAAGACAGGTTCTCTGAATCACCAGACACTGTCCATTTTGGCCGCGATGCCTGGAC	FAFJJFAJFJJJJJJJJJJJJJJJFJJJJJJJJJJJJJJJJJJJFJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJFJFJJJJJJJJJJJJJFJJJJFJJJJJJJJJJJJJJJJJJJJJJJJJFFFAA	NH:i:2	HI:i:2	AS:i:297	nM:i:0
J00153:160:HMTNMBBXX:6:1104:18893:12428	338	XM_021473592.1	1408	3	150M	*	0	0	AAGTCGACCCAAAGAGGATCTCCATCGCAGGGATGAACTCAAACACAACCAGAGGCTAAACCTGGAGGACCTGCTCAGCCACAGCTGGCTGCCCCAAGACAGGTTCTCTGAATCACCAGACACTGTCCATTTTGGCCGCGATGCCTGGAC	FAFJJFAJFJJJJJJJJJJJJJJJFJJJJJJJJJJJJJJJJJJJFJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJFJFJJJJJJJJJJJJJFJJJJFJJJJJJJJJJJJJJJJJJJJJJJJJFFFAA	NH:i:2	HI:i:2	AS:i:297	nM:i:0

One pair is mapped and both mates are present, the other pair is missing a mate completely (due to post-alignment filtering) but SAM/BAM is name sorted = the complete pair is correctly converted, the one-mate-pair read will be considered a single-end read
target/release/mudskipper bulk --alignment tests/NC_007114.7_12427_3.sam --gtf tests/NC_007114.7_12427.gtf --out test3.bam

cat <(grep -v "^@" tests/NC_007114.7_12427_3.sam) <(echo) <(samtools view test3.bam)

J00153:160:HMTNMBBXX:6:1104:18893:12428	419	NC_007114.7	24289610	3	149M	=	24289724	264	GGCAGTGGTCTCTGTCCTGGATCATGTAGAGTGTCGTTGTCAACCTGCTCCTCGCCCCGCACATCGCCGAAAGTCATCCAGCCAGAAACTAGACGCACGTGATCGTTCAGAGAAAAGTCGACCCAAAGAGGATCTCCATCGCAGGGATG	AAFFFJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJFJJFJJJJJFJJJJJJJJJJJFFJJJFJJJJJJJJJFAFAFJJJJJJJJJA<A	NH:i:2	HI:i:2	AS:i:297	nM:i:0
J00153:160:HMTNMBBXX:6:1104:18893:12428	339	NC_007114.7	24289724	3	150M	=	24289610	-264	AAGTCGACCCAAAGAGGATCTCCATCGCAGGGATGAACTCAAACACAACCAGAGGCTAAACCTGGAGGACCTGCTCAGCCACAGCTGGCTGCCCCAAGACAGGTTCTCTGAATCACCAGACACTGTCCATTTTGGCCGCGATGCCTGGAC	FAFJJFAJFJJJJJJJJJJJJJJJFJJJJJJJJJJJJJJJJJJJFJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJFJFJJJJJJJJJJJJJFJJJJFJJJJJJJJJJJJJJJJJJJJJJJJJFFFAA	NH:i:2	HI:i:2	AS:i:297	nM:i:0
J00153:160:HMTNMBBXX:6:1104:18893:12427	339	NC_007114.7	24289724	3	150M	=	24289610	-264	AAGTCGACCCAAAGAGGATCTCCATCGCAGGGATGAACTCAAACACAACCAGAGGCTAAACCTGGAGGACCTGCTCAGCCACAGCTGGCTGCCCCAAGACAGGTTCTCTGAATCACCAGACACTGTCCATTTTGGCCGCGATGCCTGGAC	FAFJJFAJFJJJJJJJJJJJJJJJFJJJJJJJJJJJJJJJJJJJFJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJFJFJJJJJJJJJJJJJFJJJJFJJJJJJJJJJJJJJJJJJJJJJJJJFFFAA	NH:i:2	HI:i:2	AS:i:297	nM:i:0

J00153:160:HMTNMBBXX:6:1104:18893:12428	419	XM_021473592.1	1294	3	149M	=	1408	264	GGCAGTGGTCTCTGTCCTGGATCATGTAGAGTGTCGTTGTCAACCTGCTCCTCGCCCCGCACATCGCCGAAAGTCATCCAGCCAGAAACTAGACGCACGTGATCGTTCAGAGAAAAGTCGACCCAAAGAGGATCTCCATCGCAGGGATG	AAFFFJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJFJJFJJJJJFJJJJJJJJJJJFFJJJFJJJJJJJJJFAFAFJJJJJJJJJA<A	NH:i:2	HI:i:2	AS:i:297	nM:i:0
J00153:160:HMTNMBBXX:6:1104:18893:12428	339	XM_021473592.1	1408	3	150M	=	1294	-264	AAGTCGACCCAAAGAGGATCTCCATCGCAGGGATGAACTCAAACACAACCAGAGGCTAAACCTGGAGGACCTGCTCAGCCACAGCTGGCTGCCCCAAGACAGGTTCTCTGAATCACCAGACACTGTCCATTTTGGCCGCGATGCCTGGAC	FAFJJFAJFJJJJJJJJJJJJJJJFJJJJJJJJJJJJJJJJJJJFJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJFJFJJJJJJJJJJJJJFJJJJFJJJJJJJJJJJJJJJJJJJJJJJJJFFFAA	NH:i:2	HI:i:2	AS:i:297	nM:i:0
J00153:160:HMTNMBBXX:6:1104:18893:12427	338	XM_021473592.1	1408	3	150M	*	0	0	AAGTCGACCCAAAGAGGATCTCCATCGCAGGGATGAACTCAAACACAACCAGAGGCTAAACCTGGAGGACCTGCTCAGCCACAGCTGGCTGCCCCAAGACAGGTTCTCTGAATCACCAGACACTGTCCATTTTGGCCGCGATGCCTGGAC	FAFJJFAJFJJJJJJJJJJJJJJJFJJJJJJJJJJJJJJJJJJJFJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJFJFJJJJJJJJJJJJJFJJJJFJJJJJJJJJJJJJJJJJJJJJJJJJFFFAA	NH:i:2	HI:i:2	AS:i:297	nM:i:0

produce warning not error, like OceanGenomics#29

Update query_bam_records.rs

993320b

janoppelt mentioned this pull request Jun 10, 2022

'main' panicked at 'index out of bounds: the len is 1 but the index is 1' #24

Open

jelber2 added 2 commits June 20, 2022 10:43

Merge branch 'OceanGenomics:main' into main

3e342ca

Update query_bam_records.rs

6e5b94d

jelber2 added 4 commits June 20, 2022 10:57

Create NC_007114.7_12427_1.sam

4e10bf8

Create NC_007114.7_12427_2.sam

5822a96

Create NC_007114.7_12427_3.sam

53e237e

Update NC_007114.7_12427_3.sam

b3cff7f

jelber2 added 2 commits June 21, 2022 09:16

Update convert.rs

4f9191b

Update query_bam_records.rs

193cd73

rob-p mentioned this pull request Jun 24, 2022

fix issue #24 #29

Merged

produce warning of missing mate

bac531a

produce warning not error, like OceanGenomics#29

thom-OG requested a review from rob-p July 3, 2024 15:45

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