Problem With Sam To Bam Converison After Alignment Of Scaffolds To Reference

Question

[Rohit]

Hello.

I'm trying to align the denovo assembly scaffolds I have generated to the available reference genome. For this I have tried using bwasw and the alignment runs fine. But I have a problem when I use samtools to convert the sam files into bam inorder to check the statistics.

I always get the following error with the commands

***[samopen] SAM header is present: 26 sequences.***
***Parse error at line 27: sequence and quality are inconsistent***
***Aborted (core dumped)***

1) samtools import Pan_troglodytes.fa.fai ref_aln_scaf.sam ref_aln_scaf.bam

2) samtools view -bS ref_aln_scaf.sam ref_aln_scaf.bam

My sam file looks like this -

scaffold1    4    *    0    0    *    *    0    0    55278387   0          -   100     #DOWN 68667525:5:56 68667525   127        +   556     #DOWN 63995574:5:107 60040051:2:45     #UP 64591229:1:67 55278387:5:56 63995574   761        -   197     #DOWN 56702323:1:-16     #UP 68667525:5:107 60040051:2:-1     *
scaffold2    4    *    0    0    *    *    0    0    55278447   0          -   100     #DOWN 67543234:3:73     #UP 61919621:2:27 67543234   144        +   405     #DOWN 50747758:4:94 48984289:1:7     #UP 55278447:3:73 50747758   614        -   61     #UP 67543234:4:94 49120931:1:131     *
scaffold3    4    *    0    0    *    *    0    0    55278537   0          +   100     #DOWN 58208681:4:63 52225285:1:127     #UP 51228731:1:62 58208681   134        -   104     #UP 63945980:1:-21 55278537:4:63     *
scaffold4    4    *    0    0    *    *    0    0    55278605   0          -   100     #DOWN 65985219:3:2     #UP 64872087:1:3 65985219   73         +   279     #DOWN 61387005:4:56 49180224:1:145     #UP 64872087:1:98 61790643:1:-22 55278605:3:2 61387005   379        +   147     #DOWN 53307077:2:155 50718052:1:26 49180224:1:61 48106164:1:112     #UP 65985219:4:56     *
scaffold5    4    *    0    0    *    *    0    0    55278615   0          +   100     #DOWN 60423174:3:74     #UP 47914359:1:129 60423174   145        +   131     #DOWN 66863269:1:15     #UP 55278615:3:74 47472933:1:118     *

I have tried using the -t option to include index too while converting but the same error is displayed. I have tried the following commands too -

samtools view -bt Pan_troglodytes.fa.fai -S ref_aln_scaf.sam -o ref_aln_scaf.bam
samtools view -bt Pan_troglodytes.fa.fai ref_aln_scaf.sam > ref_aln_scaf.bam

What is the problem here?

[Istvan Albert]

It says that the sequence and quality are inconsistent. So check the file for line 27, the quality string is and sequence seems to not match

[Rohit]

The problem is I don't have any quality... I aligned fasta sequences only against the reference

[Istvan Albert]

you would need to have a star in the quality column, do you have that?

[Rohit]

Really sorry for the late reply Istvan (crashed again)... The 5th and the 11th column for the scaffold alignment has a 0, do I replace the whole 11th column with a star???

I replaced the 11th column with a star and now I have another error

[sam_read1] reference 'scaffold3 4 * 0 0 * * 0 0 55278537 * + 100 #DOWN 58208681:4:63 52225285:1:127 #UP 51228731:1:62 58208681 134 - 104 #UP 63945980:1:-21 55278537:4:63 *' is recognized as '*'.
Parse error at line 27: invalid CIGAR character

[Devon Ryan]

You might want to post the commands you used to generate the "SAM" files, since they aren't in SAM format...

[Rohit]

I must say, now I feel really stupid

bwa index -p Pan_tro_bwaidx -a bwtsw Pan_troglodytes.fa

and - 1) bwa bwasw Pan_tro_bwaidx clint_79_k29.scaf > ref_aln_scaf.sam

[2nd try] 2) bwa bwasw clint_79_k29.scaf Pan_troglodytes.fa -f aln_ref

[Devon Ryan]

Try:

bwa bwasw Pan_troglodytes.fa clint_79_k29.scaf > ref_aln_scaf.sam

[Rohit]

I tried the command above...When I ran sam tools samtools view -bS ref_aln_scaf.sam > ref_aln_scaf.bam

I end up with the inconsistent quality error again. When I replace the 11th column of "0"s to "*"s, then tried to convert to bam, I end up with the invalid CIGAR character error again...

This seems to be a vicious cycle :(

[Devon Ryan]

Please update your original post with an excerpt of the SAM file.

[Istvan Albert]

It is not a cycle - the CIGAR string is also invalid, perhaps your entire SAM file is.

Post a few lines of the SAM file (after the header)

[Istvan Albert]

Your SAM file is invalid, what is with all those #DOWN #UP words?

[Rohit]

I updated the Sam file with the lines following the headers. I thought #UP and #DOWN words were from the sam output.

[Istvan Albert]

This does not seem to be a valid SAM file (see the SAM documentation)

QNAME   FLAG  RNAME  POS    MAPQ    CIGAR  RNEXT  TNEXT  SEQ      QUAL     ?   ?    ?
scaffold1 4    *      0      0       *      *       0     0     55278387   0   -   100

[Devon Ryan]

Nope, those have no meaning in a SAM file. What you updated with isn't SAM, I really haven't a clue how you got them.