After sorting bam file not able to index ?

Question

[sunnykevin97]

Hi after sorting not able to index the bam file showing error, how to sort it out ?

samtools index -b -@ 4 A-DS1546_1__R1_dedup_2_ReadGroupssorted.bam

[E::hts_idx_push] unsorted positions on sequence #1: 1 followed by 0 samtools index: failed to create index for "A-DS1546_1__R1_dedup_2_ReadGroupssorted.bam"

thanks!

[Bastien Hervé]

As said finswimmer in this post : How to specify the sort based on name in samtools sort?

you can only index the bam file when it ist coordinate-sorted

[sunnykevin97]

I tried to index the sorted bam file

[Bastien Hervé]

Hello sunnykevin97,

Please use the formatting bar (especially the code option) to present your post better. I've done it for you this time.

Thank you!

[ATpoint]

Can you show the sort command?

[sunnykevin97]

samtools sort -@ 4 A-DS1546_1__R1_dedup_2_ReadGroups.bam -o A-DS1546_1__R1_dedup_2_ReadGroupssorted.bam

[Bastien Hervé]

Do you have information in your A-DS1546_1__R1_dedup_2_ReadGroupssorted.bam (not empty)

[sunnykevin97]

It generated a sorted bam file ~70 GB

[Bastien Hervé]

I shorten your comment as you reply the exact same thing to ATpoint and he has took his time to reformat your comment

This is a sam file not a bam file

[ATpoint]

Come on, show some effort. Did you try anything, did you look at the data? Please show some lines of the file.

[sunnykevin97]

samtools view A-DS1546_1__R1_dedup_2_ReadGroupssorted.bam | head

C0V63ACXX120821:6:2101:1050:8813    73  chr1    0   0   101M    =   0   0   NTTCCCGTGGGGGTGTGGCTAGGCGAGACGTCATGAGCTACACTTGGAGTGTGTGCTTGATGCCAGCTCTCTTTGATCAGCGATGATTTAGAAGGCGTATT   #1=BDFFDFHHHH:@FDHGIIIIAGBA/;ECHHEHAEDEFDBDEEEC;>(;;@?A@AC<<3>A::A?ACCCCCCCACCACCB5A@>:BD>ACCCBBB9>@>   X0:i:3  X1:i:5  MD:Z:0  XG:i:0  AM:i:0  NM:i:0  SM:i:0  XM:i:3  XN:i:100    XO:i:0  XT:A:N  RG:Z:A-DS1546_1__R1
C0V4CACXX120821:1:1107:15183:40205  89  chr1    0   0   101M    =   0   0   ATTTCCCGTGGGGGTTGGGCTAGGCGAGACGTCATGAGCTACACTTGGAGTGTGTGCTTGATGCCAGCTCTCTTTGATCAGCGATGATTTAGAAGGCGTAT   ###########################################AEGHCFB@FAD?B@BB9B3:)?:31C?919@@GF?8<<GHED>IIDFBHHDAABA;;?   X0:i:3  X1:i:0  XA:Z:chrX,-51621000,101M,5;gi|117937807|gb|DQ188829.2|,+142,101M,5; MD:Z:0  XG:i:0  AM:i:0  NM:i:0  SM:i:0  XM:i:5  XN:i:100    XO:i:0  XT:A:N  RG:Z:A-DS1546_1__R1
C0V4CACXX120821:1:2202:17464:95285  163 chr1    3000001 29  15S20M1I46M19S  =   3000233 333 ATAATCTGTGTCACCTTTCCGAGGCTACCACTTCCAAGGACTACAGGAGTCGTCTCAGGTGAAAGACCATACGAAAGAGGCCCGGTTCGTACGGTAAAACA   ;@@ADD?DF4ADDBE<GGCHG<C1E=<FHFDC?DBBB3;BD;?/9B9DGHCAFFDDDDA7@.??E>DE@C@3;(6'9A?######################   MD:Z:21C33A10   XG:i:1  AM:i:29 NM:i:3  SM:i:29 XM:i:2  XO:i:1  XT:A:M  RG:Z:A-DS1546_1__R1
C0V4CACXX120821:1:2316:15160:19446  99  chr1    3000001 29  3S20M1I76M1S    =   3000256 356 ACCTTTCCGAGGCTACCACTTCCAACGACTACAGGAGTCGTCTCAGGTGAAAGACCATAAGAAAGAGGCCTGGTGTTTATGTATAAAAAGGTAATTATTAA   @@@DFFFFH>FADGIJJGGGIHHEHGIEHGGJIJIDIBFGFHEHIGG8@GHCIIHHHHHC?>CCDC>?ABDDD;?9?CC<>>@CCACCCBD>C::>@@###   MD:Z:96 XG:i:1  AM:i:29 NM:i:1  SM:i:29 XM:i:0  XO:i:1  XT:A:M  RG:Z:A-DS1546_1__R1
C0V63ACXX120821:6:2206:5185:69456   163 chr1    3000001 29  62S20M1I18M =   3000173 273 CTTCAACTATTTATACGATGTACCAATGAACACCTTCATCAAGCTCGATAATCTGTGTCACCTTTCCGAGGCTACCACTTCCAACGACTACAGGAGTCGTC   CCCFFFFFHHHHHJJJJIJJHHIJJJIIJJJJJJJJJJJJIJJJIJJJJFHIIJJIJJJJJJIJJJJIJJHFFFEECEEEDDCDDDDDDDDDDDDD?CBB?   MD:Z:38 XG:i:1  AM:i:29 NM:i:1  SM:i:29 XM:i:0  XO:i:1  XT:A:M  RG:Z:A-DS1546_1__R1
C0V63ACXX120821:7:1106:4563:28548   163 chr1    3000001 29  31S20M1I14M35S  =   3000035 135 ACCTTCATCAAGCTCGATAATCTGTGTCACCTTTCCGAGGCTACCACTTCCAACGACTACAGGAGTCGTCTCAGGTGAAAGACCATAAGAAAGAGGCCTGG   CCCFFFFFGHHHHJJJJJJJJJJGGGIJJJJJJJJJJJIJJGIIJGIIIJGHIGIIJJHFHHFFFEFDDDDDEDD@CDACDDDCDDCDDCDDCDDDDDCDB   MD:Z:34 XG:i:1  AM:i:29 NM:i:1  SM:i:29 XM:i:0  XO:i:1  XT:A:M  RG:Z:A-DS1546_1__R1
C0V63ACXX120821:7:2206:5222:58210   163 chr1    3000001 29  62S20M1I18M =   3000225 325 CTTCAACTATTTATACGATGTACCAATGAACACCTTCATCAAGCTCGATAATCTGTGTCACATTTCCGAGGCTACCACTTCCAAGGACTACAGGAGTCGTC   @@<+B?DDHHGAHIIIBGC@CCFHJHGFGHGGIJJJIGIAHFHBGIIG@FD<FH@<FBFFF).8CDF=BEED??CECEECCCAC>=?A:?CCA<A######   MD:Z:21C16  XG:i:1  AM:i:29 NM:i:2  SM:i:29 XM:i:1  XO:i:1  XT:A:M  RG:Z:A-DS1546_1__R1
C0V63ACXX120821:7:2208:17232:6944   99  chr1    3000001 29  26S20M1I54M =   3000209 309 CATCAAGCTCGATAATCTGTGTCACCTTTCCGAGGCTACCACTTCCAAGGACTACAGGAGTCGTCTCAGGTGAAAGACCATAAGAAAGAGTCCTGGTGTTT   CCCFFFFFHHHHHJJJJJFHGHJJJJJJJJIHFHGIJJJJJJJJJJIGHIIIIEHHJIGICEHBFFFFEE>ACEC>CDDDDDDDDDDDDC@CDDDD3<<@<   MD:Z:21C41G10   XG:i:1  AM:i:29 NM:i:3  SM:i:29 XM:i:2  XO:i:1  XT:A:M  RG:Z:A-DS1546_1__R1
C0V63ACXX120821:7:2210:18879:53607  99  chr1    3000001 29  59S20M1I21M =   3000217 317 CAACTATTTATACGATGTACCAATGAACACCTTCATCAAGCTCGATAATCTGTGTCACCTTTCCGAGGCTACCACTTCCAACGACTACAGGAGTCGTCTCA   @@@DDDDDHA?DFD7CB3AFAHFB3A<+A;;@GG@<EDDEHICE)0B@F<BBBDHCDBAFEGB=@AA=E?D);;36;>C:>CBBB/':A>3(23?<22??:   MD:Z:41 XG:i:1  AM:i:29 NM:i:1  SM:i:29 XM:i:0  XO:i:1  XT:A:M  RG:Z:A-DS1546_1__R1
C0V63ACXX120821:7:2309:11600:84812  163 chr1    3000004 60  17M1I83M    =   3000299 396 CCGAGGCTACCACTTCCAACGACTACAGGAGTCGTCTCAGGTGAAAGACCATAAGAAAGAGGCCTGGTGTTTATGTATAAAAAGGTAATTATTAATAAGTT   @CCFFFFDHHGFHJJJJIIIJJJIJJJJJ@FGIHHJJJJJJFHEGIJIJIEGHIJJJHFEHBFFFDD;A=?CCDDDFDDDDDDDD3>CDDEDDCCDDCAD:   X0:i:1  X1:i:0  MD:Z:93G6   XG:i:1  AM:i:37 NM:i:2  SM:i:37 XM:i:1  XO:i:1  XT:A:U  RG:Z:A-DS1546_1__R1

[swbarnes2]

Folks, is it normal for those first two entries to not have the -f 4 flagged, but to have mapping coordinates of 0?

[Bastien Hervé]

Seems like the mate is unmapped for the two first entries. But if the read is mapped there should has be a position

Can we see the alignment command line please ?

[sunnykevin97]

hello, this the command line they used..

@SQ SN:gi|117937807|gb|DQ188829.2|  LN:17010
@RG ID:A-DS1546_1__R1   PL:Illumina PU:01   LB:Library  SM:DS1546
@PG ID:bwa  PN:bwa  VN:0.7.8-r455   CL:/groups/reich/sw/bwa-0.7.8/bwa sampe /groups/reich/reference-genomes/loxAfr3_mod_mt/bwa-0.7.8/loxAfr3com.v2.fa aln.sai1 aln.sai2 R1.trim.fastq.gz R2.trim.fastq.gz

[Bastien Hervé]

Could you check if your bam is not truncated or corrupt

How to systematically check if a bam file is truncated

[shawn.w.foley]

How was the unsorted bam file generated? Is this the output from an aligner or is it converted from a sam file? Check to confirm that there is a header on your sam file:

samtools view -H sortedBamFile.bam

[sunnykevin97]

For alignment BWA sampe they used, I downloaded 7 sorted bam files from a NCBI bioproject. I don't have any problem with other bam files except this. I tried to call variants using this bam file the problems persists repeatedly. How to sort it out ?

Bam file before sorting

samtools view -H A-DS1546_1__R1_dedup_2_ReadGroups.bam | head

@HD VN:1.0  SO:coordinate
@SQ SN:chr1 LN:214701375
@SQ SN:chr10    LN:103745917
@SQ SN:chr11    LN:68864707
@SQ SN:chr12    LN:83603808
@SQ SN:chr13    LN:103893473
@SQ SN:chr14    LN:70853943
@SQ SN:chr15    LN:88640724
@SQ SN:chr16    LN:45784276
@SQ SN:chr17    LN:69133140

Bam file after sorting

samtools view -H A-DS1546_1__R1_dedup_2_ReadGroupssorted.bam | head

@HD VN:1.0  SO:coordinate
@SQ SN:chr1 LN:214701375
@SQ SN:chr10    LN:103745917
@SQ SN:chr11    LN:68864707
@SQ SN:chr12    LN:83603808
@SQ SN:chr13    LN:103893473
@SQ SN:chr14    LN:70853943
@SQ SN:chr15    LN:88640724
@SQ SN:chr16    LN:45784276
@SQ SN:chr17    LN:69133140