**30**wrote:

I'm working through `csaw`

for paired-end ATAC-seq data (see my other post for more background on my general workflow), and I don't think my BAM files are being properly read into R. I have used numerous files that are coordinate-sorted (also have tried name-sorted) and indexed (via either `samtools`

or `Rsamtools`

) and I keep getting these `Htslib`

errors and warnings upon `csaw::windowCounts`

or `csaw::regionCounts`

:

```
[W::bam_hdr_read] bgzf_check_EOF: No such file or directory
[E::bgzf_read] Read block operation failed with error -1 after zd of zu bytes
```

As the command continues, the first warning appears many times. The files ultimately end up loading, but I believe they are only partially read, as none of my `macs2`

peaks on chr1 show up (0 counts across all samples) and chrX and chrY do not appear to be read at all.

Example for

```
> standard.chr <- paste0("chr", c(1:19, "X", "Y"))
> param <- readParam(max.frag=1000, pe="both",
discard=blacklist, restrict=standard.chr)
> binned <- windowCounts(pe.bams, bin=TRUE, width=10000, param=param)
> binned@rowRanges@seqnames
factor-Rle of length 211698 with 19 runs
Lengths: 4 12711 11876 11694 11717 12151 10069 9504 9176 8749 5822 17839 15645 15223 14806 14637 14194 12570 3311
values : chr1 chr10 chr11 chr12 chr13 chr14 chr15 chr16 chr17 chr18 chr19 chr2 chr3 chr4 chr5 chr6 chr7 chr8 chr9
Levels(21): chr1 chr10 chr11 chr12 chr13 chr14 chr15 chr16 chr17 chr18 ... chr3 chr4 chr5 chr6 chr7 chr8 chr9 chrX chrY
```

As you can see, no 10kb bins were called for chrX or chrY, and only 4 were called for chr1.

I'm pretty certain the BAM file is not corrupted as the headers and EOF exist, and `samtools quickcheck`

elicits no errors.

Example of header via `samtools view -H file.sorted.bam`

```
@HD VN:1.5 SO:coordinate
@SQ SN:chr1 LN:195471971
@SQ SN:chr10 LN:130694993
@SQ SN:chr11 LN:122082543
@SQ SN:chr12 LN:120129022
@SQ SN:chr13 LN:120421639
@SQ SN:chr14 LN:124902244
@SQ SN:chr15 LN:104043685
@SQ SN:chr16 LN:98207768
@SQ SN:chr17 LN:94987271
@SQ SN:chr18 LN:90702639
@SQ SN:chr19 LN:61431566
@SQ SN:chr1_GL456210_random LN:169725
@SQ SN:chr1_GL456211_random LN:241735
@SQ SN:chr1_GL456212_random LN:153618
@SQ SN:chr1_GL456213_random LN:39340
@SQ SN:chr1_GL456221_random LN:206961
@SQ SN:chr2 LN:182113224
@SQ SN:chr3 LN:160039680
@SQ SN:chr4 LN:156508116
@SQ SN:chr4_GL456216_random LN:66673
@SQ SN:chr4_JH584292_random LN:14945
@SQ SN:chr4_GL456350_random LN:227966
@SQ SN:chr4_JH584293_random LN:207968
@SQ SN:chr4_JH584294_random LN:191905
@SQ SN:chr4_JH584295_random LN:1976
@SQ SN:chr5 LN:151834684
@SQ SN:chr5_JH584296_random LN:199368
@SQ SN:chr5_JH584297_random LN:205776
@SQ SN:chr5_JH584298_random LN:184189
@SQ SN:chr5_GL456354_random LN:195993
@SQ SN:chr5_JH584299_random LN:953012
@SQ SN:chr6 LN:149736546
@SQ SN:chr7 LN:145441459
@SQ SN:chr7_GL456219_random LN:175968
@SQ SN:chr8 LN:129401213
@SQ SN:chr9 LN:124595110
@SQ SN:chrX LN:171031299
@SQ SN:chrX_GL456233_random LN:336933
@SQ SN:chrY LN:91744698
@SQ SN:chrY_JH584300_random LN:182347
@SQ SN:chrY_JH584301_random LN:259875
@SQ SN:chrY_JH584302_random LN:155838
@SQ SN:chrY_JH584303_random LN:158099
@SQ SN:chrUn_GL456239 LN:40056
@SQ SN:chrUn_GL456367 LN:42057
@SQ SN:chrUn_GL456378 LN:31602
@SQ SN:chrUn_GL456381 LN:25871
@SQ SN:chrUn_GL456382 LN:23158
@SQ SN:chrUn_GL456383 LN:38659
@SQ SN:chrUn_GL456385 LN:35240
@SQ SN:chrUn_GL456390 LN:24668
@SQ SN:chrUn_GL456392 LN:23629
@SQ SN:chrUn_GL456393 LN:55711
@SQ SN:chrUn_GL456394 LN:24323
@SQ SN:chrUn_GL456359 LN:22974
@SQ SN:chrUn_GL456360 LN:31704
@SQ SN:chrUn_GL456396 LN:21240
@SQ SN:chrUn_GL456372 LN:28664
@SQ SN:chrUn_GL456387 LN:24685
@SQ SN:chrUn_GL456389 LN:28772
@SQ SN:chrUn_GL456370 LN:26764
@SQ SN:chrUn_GL456379 LN:72385
@SQ SN:chrUn_GL456366 LN:47073
@SQ SN:chrUn_GL456368 LN:20208
@SQ SN:chrUn_JH584304 LN:114452
@PG ID:bowtie2 PN:bowtie2 VN:2.2.6 ...
# rest of header...
```

And here's an example read via `samtools view file.sorted.bam | head`

:

```
NS500653:255:H2YWNAFXY:3:11506:4441:16945 99 chr1 3000222 27 75M = 3000302 15 GTCTAGGAAGTTGTCCATTTCATCCAGGTTTTCCTGGTTTTTTTTTAGTATAGCCTTTCATAGTAGAATCTGATG AAAAAEEEEEEEEEEEEEEEEEEEEEEEEEEEEAEEEEEEEEEEEE/EEEEEAEAEEEEAE/E<A<AAA6EEEEA MD:Z:65A9 PG:Z:MarkDuplicates XG:i:0 NM:i:1 XM:i:1 XN:i:0 XO:i:0 AS:i:-4 XS:i:-41 YS:i:-5YT:Z:CP
```

Any suggestions for how to fix these issues when loading BAMs into `csaw`

? I'm working on Windows, so maybe it would help to try this out on the Centos cluster.

**0**• written 2.6 years ago by reskejak •

**30**