[maubp]

samtools doesn't work at all, probably something wrong with the installation. I downloaded it and extracted the file, so now i have a file :samtools-0.1.18 with many files inside. But when i tried to run samtools I recieve: bash: samtools: command not found
How this can be fixed?

You don't just download and unzip samtools - you must compile it. What OS are you on?

[litali]

samtools

i use linux redhat

[maubp]

i use linux redhat

Then the installation shouldn't be a problem - follow the instructions in the INSTALL file.

[litali]

Igv

ok, I installed samtools and created the sorted and the index files. Now I tried to upload them into the IGV viewer, but still I see no alignments..

[ulz_peter]

does it say zoom in to view alignments or is it just empty?

[maubp]

Progress. I'm now repeating myself but:

What does "samtools idxstats" tell you? This should summarise how many reads were mapped - perhaps very few mapped.

Also the first few lines in SAM format would be interesting for diagnosis, try "samtools view example.bam | head -n 50" for the first 50 lines. If you post them here, wrap it with [ code ] and [ /code ] tags to get it to display nicely on the forum.

[litali]

IGv

it says: " loading SAM/BAM index files is not supported ....sorted.bam.bai..Load the SAM/ BAM files directly"
When I click "ok" it says "xoom in to see the alignments" but even if I soom in, it is empty

[maubp]

it says: " loading SAM/BAM index files is not supported ....sorted.bam.bai..Load the SAM/ BAM files directly"
When I click "ok" it says "xoom in to see the alignments" but even if I soom in, it is empty

i.e. If you open the BAM file with IGV (or another viewer like Tablet), it should automatically find and use the index file (named *.bam.bai or *.bai).

It does sound like there is a problem with the BAM file from Newbler - which is why I was suggesting looking at the file with samtools idxstats etc.

[litali]

samtools, igv and more

when i try samtools idxstats, I receive command not found. indeed in the help menu of samtools which I installed I have only:
Program: samtools (Tools for alignments in the SAM format)
Version: 0.1.6 (r453)

Usage: samtools <command> [options]

Command: view SAM<->BAM conversion
sort sort alignment file
pileup generate pileup output
faidx index/extract FASTA
tview text alignment viewer
index index alignment
fixmate fix mate information
glfview print GLFv3 file
flagstat simple stats
calmd recalculate MD/NM tags and '=' bases
merge merge sorted alignments (Picard recommended)
rmdup remove PCR duplicates (Picard recommended)
maybe it is because this is an old version?
However, when I look at the first lines of the file as you suggested, I receive:
[ code ]
F01BJ5E01C2F11 16 chrX 138807104 100 16M1I6M1D3M1I54M1I1D104ATCTATTATGTATCAAATAAAAATAAATAAAATATTGTTCACTATTTTTTCTAGTGATAACTCAAACTAGAATCCAAAACTGCTAACAATATACTAAAGGTCAACTTCAGTGAAAAGTGAATTGGGCCGAGCGCAGAGGCTCACACCTGTAATCCCAGCACTTTGGGAAGCCGAGGCGGGCGGACA ***622....26644...(4......47======>??>6222==9:::;::1111;====5566:===8;111167=:777;;;==;77788=@?????????AABAA????AA??????A@????????><9979<<997779979==<46:1106886:<562222285888::8:9822222;
F01BJ5E01DMYWU 0 chrX 138807511 100 12M1I2M1I159M1D50M A AAAAAGAAAAAAGTAGAATTGTTTACTTTCTGTGCACCTTTTCTACTGGGACAGTCTTTCTGAGAAGTGCTTTAAGTGCATGTTAGAAGCCAGGGACATTTAGCCAGGCGTGGTGGCACGTGACTGTAGTCCCAGCTACTCAGGAGGCTGAGGTGGGAGGATCTCTTGAACGGGAAGTCAAGGCTGCAGTGAGCTATGATCATGCCACTGCACTCCAGCCTGGG C@@<<<@@CCCC@@@A@>>>@A43//////6*222--AA-@@@==<000<>---4/64<<<>>////3-<<<<<<<<<<AAA<3304;????:9:ABB444156@CCCCAAACAAAAAAAAAAA@@?AAACCCCCCCCCCCCCCCCCCAAACCA?@@CCCCCCCCCCCCCCCCCCCCCCCC?:77CCB>9999>>92222259AAAAAACCCCCCCCCCCCCCC@
[ /code]
etc...
In the IGV if I upload the bam file only it says @zoom in to see the alignments@ but it is empty...

[maubp]

when i try samtools idxstats, I receive command not found. indeed in the help menu of samtools which I installed I have only:
Program: samtools (Tools for alignments in the SAM format)
Version: 0.1.6 (r453)
...

That is very out of date, the current release is samtools 0.1.18.

P.S. Once you have updated samtools, rebuild the index. The newer versions include more information in the index which is used by samtools idxstats and some viewers too.

P.P.S. You need to leave the spaces out of the [ code ] and the closing tag [ /code ] - I'm using them here otherwise you wouldn't see them, just their formatting effect.

UPDATE - see end

However, there does seem to be a problem with this 1st read - despite columns 3 to 5 saying it maps to chrX at position 138807104 with quality 100, the FLAG in column 2 is 16 (0x010 in hex) and that says reverse complemented but not mapped.

F01BJ5E01C2F11 16 chrX 138807104 100 16M1I6M1D3M1I54M1I1D104 ATCTATTATGTATCAAATAAAAATAAATAAAATATTGTTCACTATTTTTTCTAGTGATAACTCAAACTAGAATCCAAAACTGCTAACAATATACTAAGGTCAACTTCAGTGAAAAGTGAATTGGGCCGAGCGCAGAGGCTCACACCTGTAATCCCAGCACTTTGGGAAGCCGAGGCGGGCGGACA ***622....26644...(4......47======>??>6222==9:::;::1111;====5566:===8;111167=:777;;;==;77788=@?????????AABAA????AA??????A@????????><9979<<997779979==<46:1106886:<562222285888::8:9822222;

The second read is similar, here the flag is just 0 meaning not mapped (and not reverse complemented):

F01BJ5E01DMYWU 0 chrX 138807511 100 12M1I2M1I159M1D50M AAAAAAGAAAAAAGTAGAATTGTTTACTTTCTGTGCACCTTTTCTACTGGGACAGTCTTTCTGAGAAGTGCTTTAAGTGCATGTTAGAAGCCAGGGACATTTAGCCAGGCGTGGTGGCACGTGACTGTAGTCCCAGCTACTCAGGAGGCTGAGGTGGGAGGATCTCTTGAACGGGAAGTCAAGGCTGCAGTGAGCTATGATCATGCCACTGCACTCCAGCCTGGG C@@<<<@@CCCC@@@A@>>>@A43//////6*222--AA-@@@==<000<>---4/64<<<>>////3-<<<<<<<<<<AAA<3304;????:9:ABB444156@CCCCAAACAAAAAAAAAAA@@?AAACCCCCCCCCCCCCCCCCCAAACCA?@@CCCCCCCCCCCCCCCCCCCCCCCC?:77CCB>9999>>92222259AAAAAACCCCCCCCCCCCCCC@

This looks like a problem with the BAM output from Newbler gsMapper v2.6, but having only looked at two reads it is premature to say that for sure.

UPDATE - Apologies, I miss read the FLAG. The mapping (or not) is set in the FLAG as bit 0x4, but I had the meaning backwards. FLAGs of 0 and 16 are mapped (forward and reverse strand). So you should see something on chrX if you zoom in.

[litali]

Bam

ok, i installed the newer version of samtools, so idxstats gives the following:
chrX 154913754 47196 0
* 0 0 0

I also attach a few more reads from the first lines of the sam format:

F01BJ5E01DBVMG  0       chrX    138807718       100     1M1D34M1I2M1I10M1I6M1I1M1I1M1D1M1I81M1I10M1I1M1I26M1I17M        *       0       0       CGCACTCCAGCCTGGGCAACAGAGCAAAACCCTGTAGTACAAAAAAAAAGAAAAAAAGACGGCAGCAGCCAGGGATATGAATTAGGAGTGGGGTGGGTAGAGAGTGAGTGGGGCTGCTGGAGACAATGTTCCCATGGCACTGAACCCTGGTTAAACCAGTCTTTGAGCAAGTACTATCACTTGTCTGTAATTCCTTCTTCC       =922229;=9::<<<====;:8000308686633223000388658003..55:::82020602.........2'''''''0'866600009966000069996600000034.....---3.,.3...783...334...444473......97.......474.......44.......+444....434433----38
F01BJ5E01C26ZG  0       chrX    138807757       100     3M10D96M1D1I118M1I2M   AAAAAGCAGCAGCAGCCAGGGATATGAATTAGGAGTGGGGTGGGTAGAGAGTGAGTGGGGCTGCTGGAGACAATGTTCCCATGGCACTGACCCTGGTTAGCAGTCTTTGAGCAAGTACTATCACTTGCTGTAATTCCTTCTTCCTCATCCTTTGCTCCTTTTGAATATGATGATTTCTAGGAATGAACCTTCTTTATGACACATGCTGTATATTATTTTGG    D=======ABDDAAA@ACCCCCCC??FA?CCCDEEEDDCCCCCCCCCCCBAAAACCCCCCCCCCC555@CCCCCCCCCCBCCCCAAABCCCCCCCCCCCCCCCCAAACCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCC@@@??CCCCCBBBBCB;:444?BBBB:::B;;;;CCB;;;@>
F01BJ5E01B5WV8  0       chrX    138807757       100     3M10D3M1I77M1D15M1D1I118M1I2M   *       0       0       AAAAAGACAGCAGCAGCCAGGGATATGAATTAGGAGTGGGGTGGGTAGAGAGTGAGTGGGGCTGCTGGAGACAATGTTCCCATGCACTGACCCTGGTTAGCAGTCTTTGAGCAAGTACTATCACTTGCTGTAATTCCTTCTTCCTCATCCTTTGCTCCTTTTGAATATGATGATTTCTAGGAATGAACCTTCTTTATGACACATGCTGTATATTATTTTGG   =922229;<9::::85888888651111.3)86<<=====<99::???<<<>>??=>>==<<<<<<+++<><??????@????<888<>><<<=:;::;=7722277777====<<<??????<<<><888:;;====66566:666:::==>??>>6677====<<<<9;:<<633468.......444234363-00::49;;;;;;A>>>>>?>>>;:
F01BJ5E01CD9VD  16      chrX    138807757       100     5M8D52M1I49M1I88M1I2M1D26M      *       0       0       AAAAAAAGCAGCAGCAGCCAGGGATATGAATTAGGAGTGGGGTGGGTAGAGAGTGAGGTGGGGCTGCTGGAGACAATGTTCCCATGGCACTGACCCTGGTTAACAGTTCTTTGAGCAAGTACTATCACTTGCTGTAATTCCTTCTTCCTCATCCTTTGCTCCTTTTGAATATGATGATTTCTAGGAATGAACCTTCCTTATGACACATGCTGTATATTATTTGGT       888<>ACCCCAA/95==59999AA>>99<9//--7////<<<770009A>>>>>AA>>>999999AAAA9::::AAAAAB@90033@@?@@@?ACCCCCA;;;;@AAA?@9;;99;;999>>??A;;043>>AA?1;;;;<<@?BBBCCCA@@<<<@A?@@@@@@@A??99<<<<AAACCCCCCCACCCC?@77=<CCCCCCCCAAAACCC@@<<;AAC<<<<<C
F01BJ5E01DMLRK  0       chrX    138807792       100     3M1I3M1I119M1I2M1D2M1I1D19M1D1M1I6M1I25M        *       0       0       GAACTTACGGAGTGGGGTGGGTAGAGAGTGAGTGGGGCTGCTGGAGACAATGTTCCCATGGCACTGACCCTGGTTAACAGTCTTTGAGCAAGTACTATCACTTGCTGTAATTCCTTCTTCCTCATCCGTTGCCCCTTTTGAATATGATGATTCCTAGGAAATGAACCTTCTTTATGACACATGCTG     ;2222299<777:<:=<:::82003220220022658556.0***080000620202....222822225:<00280000299;======???<<<<====;86000006974...83433:666664444799.............79............2......34444.............
F01BJ5E01DZ0XI  0       chrX    138807831       100     114M1I40M       *      TGGAGACAATGTTCCCATGGCACTGACCCTGGTTAACAGTCTTTGAGCAAGTACTATCACTTGCTGTAATTCCTTCTTCCTCATCCTTTGCTCCTTTTGAATATGATGATTTCTTAGGAATGAACCTTCTTTATGACACATGCTGTATATTATTT     <8222288:8::8997<<9999?=??????A?<==<?@?:::::::<??=>><>>>22229589802299===>>>=?????@?>>>>=;76666990000000====;;98899;.........9799;;99;;11..06777779944.....

I see the flag in coloumn2 is always 0 or 16, though I have the positions of the mapping,,
So, do you think it is a problem with the BAM file?
Thank you alot!!!

[maubp]

Apologies, I miss read the FLAG. The mapping (or not) is set in the FLAG as 0x4, but I had the meaning backwards. FLAGs of 0 and 16 are mapped (forward and reverse strand).

So you should see something on chrX if you zoom in - try the area where those reads are mapped, coordinate 138807718.

[litali]

Bam

Thank you alot!
I now can see the akignments in IGV. However, I think I only see the coverage. how can I see the SNPs or the reads themselves? I now see where the reads are mapped, but it is just grey lines with black dots. If I zoom more I only see the bases in the reference but not in the reads themselves

[litali]

I found hoe to see the bases, thanx!! another question: what does it mean the "cigar" line with a number following it?
and also is it possible to use noe this sorted bam file to upload it to ucsc and see it in the viewer there?

[maubp]

Thank you alot!
I now can see the akignments in IGV. However, I think I only see the coverage. how can I see the SNPs or the reads themselves? I now see where the reads are mapped, but it is just grey lines with black dots. If I zoom more I only see the bases in the reference but not in the reads themselves

OK, I guess it was the out of date index from the old version of samtools that was the problem.

By default IGV shows the reads in grey where they match the reference. I think you can right click to change the colour scheme.

Alternatively, you might find another viewer more intuitive - I like Tablet http://bioinf.hutton.ac.uk/tablet/ although currently IGV handles inserts in SAM/BAM better.

I found hoe to see the bases, thanx!! another question: what does it mean the "cigar" line with a number following it?
and also is it possible to use noe this sorted bam file to upload it to ucsc and see it in the viewer there?

The CIGAR string describes how the read maps onto the reference. The viewer will use this information to construct the display. The details (and the complicated FLAG field I was looking at earlier) are in the SAM/BAM specification:
http://samtools.sourceforge.net/SAM1.pdf