Ah I see. That is exactly what I had done... So if I am trying to create my own reference genome (I am trying to make it sex specific by hard masking the Y chromosome) would it just be best to use the STAR generate genome command where I keep each chromosome fasta file separate?
Is my output .bam file from STAR ok?
Hello!
I recently ran a sample against a reference genome and received a .bam output. The results are shown below after I used samtools view SRR6350434_STARAligned.out.bam | head:
SRR6350434.407 163 1 2009825263 0 21S86M = 2009825266 93 ATATGGATCCGGCGCGCCGTCGACTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTATTTTT AA<FFJJFJJJJJ-FJJJ-AFJJ7-F-FJJ-JJFJJ-FJJ-FJJJFJFAFJJJJJJJ<AJAAA-<-F-AJFAJJJFJJJJJJJ<<AJJ-<<JFA<JJ-7<J-<AF-A NH:i:9 HI:i:1 AS:i:168 nM:i:3
SRR6350434.407 83 1 2009825266 0 90M24S = 2009825263 -93 TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTATTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT <--F-<-JFAAJJFFAAJJJFJF<FJJJFFJJJJJFJAFAAFFFF-F-<JF7AFF-<JJJA-F7JA-FJJJAJF<JFFFJJJJJJJJJJJJJAJJJJJJJJJJJFJJJJFF<AA NH:i:9 HI:i:1 AS:i:168 nM:i:3
SRR6350434.407 419 1 2009825273 0 24S83M = 2009825266 83 ATATGGATCCGGCGCGCCGTCGACTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTATTTTT AA<FFJJFJJJJJ-FJJJ-AFJJ7-F-FJJ-JJFJJ-FJJ-FJJJFJFAFJJJJJJJ<AJAAA-<-F-AJFAJJJFJJJJJJJ<<AJJ-<<JFA<JJ-7<J-<AF-A NH:i:9 HI:i:2 AS:i:167 nM:i:2
SRR6350434.407 339 1 2009825266 0 90M24S = 2009825273 -83 TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTATTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT <--F-<-JFAAJJFFAAJJJFJF<FJJJFFJJJJJFJAFAAFFFF-F-<JF7AFF-<JJJA-F7JA-FJJJAJF<JFFFJJJJJJJJJJJJJAJJJJJJJJJJJFJJJJFF<AA NH:i:9 HI:i:2 AS:i:167 nM:i:2
SRR6350434.407 419 1 2009825272 0 24S83M = 2009825266 84 ATATGGATCCGGCGCGCCGTCGACTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTATTTTT AA<FFJJFJJJJJ-FJJJ-AFJJ7-F-FJJ-JJFJJ-FJJ-FJJJFJFAFJJJJJJJ<AJAAA-<-F-AJFAJJJFJJJJJJJ<<AJJ-<<JFA<JJ-7<J-<AF-A NH:i:9 HI:i:3 AS:i:167 nM:i:2
SRR6350434.407 339 1 2009825266 0 90M24S = 2009825272 -84 TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTATTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT <--F-<-JFAAJJFFAAJJJFJF<FJJJFFJJJJJFJAFAAFFFF-F-<JF7AFF-<JJJA-F7JA-FJJJAJF<JFFFJJJJJJJJJJJJJAJJJJJJJJJJJFJJJJFF<AA NH:i:9 HI:i:3 AS:i:167 nM:i:2
SRR6350434.407 419 1 2009825271 0 24S83M = 2009825266 85 ATATGGATCCGGCGCGCCGTCGACTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTATTTTT AA<FFJJFJJJJJ-FJJJ-AFJJ7-F-FJJ-JJFJJ-FJJ-FJJJFJFAFJJJJJJJ<AJAAA-<-F-AJFAJJJFJJJJJJJ<<AJJ-<<JFA<JJ-7<J-<AF-A NH:i:9 HI:i:4 AS:i:167 nM:i:2
SRR6350434.407 339 1 2009825266 0 90M24S = 2009825271 -85 TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTATTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT <--F-<-JFAAJJFFAAJJJFJF<FJJJFFJJJJJFJAFAAFFFF-F-<JF7AFF-<JJJA-F7JA-FJJJAJF<JFFFJJJJJJJJJJJJJAJJJJJJJJJJJFJJJJFF<AA NH:i:9 HI:i:4 AS:i:167 nM:i:2
SRR6350434.407 419 1 2009825270 0 24S83M = 2009825266 86 ATATGGATCCGGCGCGCCGTCGACTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTATTTTT AA<FFJJFJJJJJ-FJJJ-AFJJ7-F-FJJ-JJFJJ-FJJ-FJJJFJFAFJJJJJJJ<AJAAA-<-F-AJFAJJJFJJJJJJJ<<AJJ-<<JFA<JJ-7<J-<AF-A NH:i:9 HI:i:5 AS:i:167 nM:i:2
SRR6350434.407 339 1 2009825266 0 90M24S = 2009825270 -86 TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTATTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT <--F-<-JFAAJJFFAAJJJFJF<FJJJFFJJJJJFJAFAAFFFF-F-<JF7AFF-<JJJA-F7JA-FJJJAJF<JFFFJJJJJJJJJJJJJAJJJJJJJJJJJFJJJJFF<AA NH:i:9 HI:i:5 AS:i:167 nM:i:2
My end goal is to use htseq-count, so I sorted my .bam file using the command samtools sort SRR6350434_STARAligned.out.bam -o SRR6350434.sorted.bam The results using the samtools view command again resulted in the following:
SRR6350434.20382235 355 1 10005 1 86M24S = 10007 97 CCTAACCCTAACCCTAACCCTAACCCTAACCCTAACCCTAACCCTAACCCTAACCCTAACCCTAACCCTAACCCTAACCCTAACCCCTAACCCTAACCCTAACCCTAACC AAFFFJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJFJFJJJJJFJJJAFJJJJJ7F7FJF<J7JJJ7F-FJJ<F-AF NH:i:3 HI:i:2 AS:i:175 nM:i:2
SRR6350434.20382235 355 1 10005 1 86M24S = 10013 103 CCTAACCCTAACCCTAACCCTAACCCTAACCCTAACCCTAACCCTAACCCTAACCCTAACCCTAACCCTAACCCTAACCCTAACCCCTAACCCTAACCCTAACCCTAACC AAFFFJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJFJFJJJJJFJJJAFJJJJJ7F7FJF<J7JJJ7F-FJJ<F-AF NH:i:3 HI:i:3 AS:i:175 nM:i:2
SRR6350434.20382235 403 1 10007 1 95M = 10005 -97 TACCCCTACCCCTAACCCTAACCCTAACCCTAACCCTAACCCTAACCCTAACCCTAACCCTAACCCTAACCCTAACCCTAACCCTAACCCTAACC 7--FJFAA-JJA77-JJAF--JJFA<-JJJAA<JJJF7-JJJFF<JJJFJFJJJAFFJJJJJFJJJJJFJJJFJJJJJJJJJJJJJJJJJFFAAA NH:i:3 HI:i:2 AS:i:175nM:i:2
SRR6350434.20382235 99 1 10011 1 86M24S = 10013 97 CCTAACCCTAACCCTAACCCTAACCCTAACCCTAACCCTAACCCTAACCCTAACCCTAACCCTAACCCTAACCCTAACCCTAACCCCTAACCCTAACCCTAACCCTAACC AAFFFJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJFJFJJJJJFJJJAFJJJJJ7F7FJF<J7JJJ7F-FJJ<F-AF NH:i:3 HI:i:1 AS:i:175 nM:i:2
SRR6350434.20382235 147 1 10013 1 95M = 10011 -97 TACCCCTACCCCTAACCCTAACCCTAACCCTAACCCTAACCCTAACCCTAACCCTAACCCTAACCCTAACCCTAACCCTAACCCTAACCCTAACC 7--FJFAA-JJA77-JJAF--JJFA<-JJJAA<JJJF7-JJJFF<JJJFJFJJJAFFJJJJJFJJJJJFJJJFJJJJJJJJJJJJJJJJJFFAAA NH:i:3 HI:i:1 AS:i:175nM:i:2
SRR6350434.20382235 403 1 10013 1 95M = 10005 -103 TACCCCTACCCCTAACCCTAACCCTAACCCTAACCCTAACCCTAACCCTAACCCTAACCCTAACCCTAACCCTAACCCTAACCCTAACCCTAACC 7--FJFAA-JJA77-JJAF--JJFA<-JJJAA<JJJF7-JJJFF<JJJFJFJJJAFFJJJJJFJJJJJFJJJFJJJJJJJJJJJJJJJJJFFAAA NH:i:3 HI:i:3 AS:i:175nM:i:2
SRR6350434.4260755 419 1 10629 0 9M42S = 134164 123659 GGCGCGCCGTCGACTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTGCCCC AAFFFJJJJ7A<AJJAAFJ<<<FJJFJJJJJFJJFJJJFF<<JF-<<-<-7 NH:i:7 HI:i:7 AS:i:129 nM:i:0
SRR6350434.4260755 419 1 10658 0 9M42S = 134164 123630 GGCGCGCCGTCGACTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTGCCCC AAFFFJJJJ7A<AJJAAFJ<<<FJJFJJJJJFJJFJJJFF<<JF-<<-<-7 NH:i:7 HI:i:6 AS:i:129 nM:i:0
SRR6350434.4260755 419 1 10687 0 9M42S = 134164 123601 GGCGCGCCGTCGACTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTGCCCC AAFFFJJJJ7A<AJJAAFJ<<<FJJFJJJJJFJJFJJJFF<<JF-<<-<-7 NH:i:7 HI:i:5 AS:i:129 nM:i:0
SRR6350434.4260755 419 1 10716 0 9M42S = 134164 123572 GGCGCGCCGTCGACTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTGCCCC AAFFFJJJJ7A<AJJAAFJ<<<FJJFJJJJJFJJFJJJFF<<JF-<<-<-7 NH:i:7 HI:i:4 AS:i:129 nM:i:0
However when I try to index (using samtools index SRR6350434.sorted.bam) it I get the following error:
[E::hts_idx_check_range] Region 536894414..536894564 cannot be stored in a bai index. Try using a csi index
[E::sam_index] Read 'SRR6350434.8195878' with ref_name='1', ref_length=3090159160, flags=163, pos=536894415 cannot be indexed
samtools index: failed to create index for "SRR6350434.sorted.bam": Result too large
I then tried to use a csi index with samtools index -c SRR6350434.sorted.bam and received the following error:
[E::hts_idx_push] Unsorted positions on sequence #1: 2147458980 followed by -2147454349
[E::sam_index] Read 'SRR6350434.20327797' with ref_name='1', ref_length=3090159160, flags=147, pos=-2147454349 cannot be indexed
samtools index: failed to create index for "SRR6350434.sorted.bam"
Now I am wondering if there is something wrong with the .bam output from STAR.
Any insight would be greatly appreciated!
Thank you!
1 answer
is it a human genome. All the chromosome should be in a separate fasta entry (= do not concatenate all the chromosomes into a single fasta entry).
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Pierre Lindenbaum
I don't know if you can make a STAR index with lots of separate fasta files. Where did you even get a fasta where all the chromosome are concatenated together into a single sequence? (What were you going to do downstream with genomic coordinates like that?)
Aren't there regions of the X chromosome which are homologous to the Y chromosome? Aren't you going to get false mapping to the X in male samples?
If this is all new to you, all the more reason not to be cute by trying to make your own protocol.
I am only looking at female samples and therefore am only interested in an XX specific reference. I am not using my own protocol, but instead using one specifically designed for generating sex specific reference genomes (which specifically has a cat command to concatenate the sequences). I have contacted them after reading the previous comments to better understand the issue.
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what is the output of
well.. you must specify the correct path to your bam files....
Sorry about that I thought I had them in the same directory! When I run it again with the correct path it results in
all okYour reference has a single sequence which is 3 billion bases long?
I assumed that was referring to the reference genome that I used and I assumed that it would make sense for it to be 3 billion bases. Is that incorrect? Should it not be a single sequence? Sorry if that is a silly question this is all new to me.