I am running the following command:

bwa mem -t 4 /users/person/resources/reference/hg19/genome/ucsc.hg19.fasta 160095-T_S2_L003_R1_001.fastq.gz 160095-t_s2_l003_r2_001.fastq.gz > 160095-T_S2_L003_R1_001.fastq.gz.bwa.sam

With the following bwa messages where all four orientations are being skipped:-

```
[M::bwa_idx_load_from_disk] read 0 ALT contigs
[M::process] read 546760 sequences (40000002 bp)...
[M::process] read 546534 sequences (40000140 bp)...
[M::mem_pestat] # candidate unique pairs for (FF, FR, RF, RR): (1, 1, 0, 0)
[M::mem_pestat] skip orientation FF as there are not enough pairs
[M::mem_pestat] skip orientation FR as there are not enough pairs
[M::mem_pestat] skip orientation RF as there are not enough pairs
[M::mem_pestat] skip orientation RR as there are not enough pairs
[mem_sam_pe] paired reads have different names: "NS500784:187:HHYNGAFXX:4:11401:17815:1049", "NS500784:187:HHYNGAFXX:1:11101:13503:1043"
[mem_sam_pe] paired reads have different names: "NS500784:187:HHYNGAFXX:4:11401:8106:1064", "NS500784:187:HHYNGAFXX:1:11101:4077:1043"
```

When I open the sam file it looks like this:

```
@SQ SN:chrM LN:16571
@SQ SN:chr1 LN:249250621
@SQ SN:chr2 LN:243199373
@SQ SN:chr3 LN:198022430
@SQ SN:chr4 LN:191154276
@SQ SN:chr5 LN:180915260
@SQ SN:chr6 LN:171115067
@SQ SN:chr7 LN:159138663
@SQ SN:chr8 LN:146364022
@SQ SN:chr9 LN:141213431
@SQ SN:chr10 LN:135534747
@SQ SN:chr11 LN:135006516
@SQ SN:chr12 LN:133851895
@SQ SN:chr13 LN:115169878
@SQ SN:chr14 LN:107349540
@SQ SN:chr15 LN:102531392
@SQ SN:chr16 LN:90354753
@SQ SN:chr17 LN:81195210
@SQ SN:chr18 LN:78077248
@SQ SN:chr19 LN:59128983
@SQ SN:chr20 LN:63025520
@SQ SN:chr21 LN:48129895
@SQ SN:chr22 LN:51304566
@SQ SN:chrX LN:155270560
@SQ SN:chrY LN:59373566
@SQ SN:chr1_gl000191_random LN:106433
@SQ SN:chr1_gl000192_random LN:547496
@SQ SN:chr4_ctg9_hap1 LN:590426
@SQ SN:chr4_gl000193_random LN:189789
@SQ SN:chr4_gl000194_random LN:191469
@SQ SN:chr6_apd_hap1 LN:4622290
@SQ SN:chr6_cox_hap2 LN:4795371
@SQ SN:chr6_dbb_hap3 LN:4610396
@SQ SN:chr6_mann_hap4 LN:4683263
@SQ SN:chr6_mcf_hap5 LN:4833398
@SQ SN:chr6_qbl_hap6 LN:4611984
@SQ SN:chr6_ssto_hap7 LN:4928567
@SQ SN:chr7_gl000195_random LN:182896
@SQ SN:chr8_gl000196_random LN:38914
@SQ SN:chr8_gl000197_random LN:37175
@SQ SN:chr9_gl000198_random LN:90085
@SQ SN:chr9_gl000199_random LN:169874
@SQ SN:chr9_gl000200_random LN:187035
@SQ SN:chr9_gl000201_random LN:36148
@SQ SN:chr11_gl000202_random LN:40103
@SQ SN:chr17_ctg5_hap1 LN:1680828
@SQ SN:chr17_gl000203_random LN:37498
@SQ SN:chr17_gl000204_random LN:81310
@SQ SN:chr17_gl000205_random LN:174588
@SQ SN:chr17_gl000206_random LN:41001
@SQ SN:chr18_gl000207_random LN:4262
@SQ SN:chr19_gl000208_random LN:92689
@SQ SN:chr19_gl000209_random LN:159169
@SQ SN:chr21_gl000210_random LN:27682
@SQ SN:chrUn_gl000211 LN:166566
@SQ SN:chrUn_gl000212 LN:186858
@SQ SN:chrUn_gl000213 LN:164239
@SQ SN:chrUn_gl000214 LN:137718
@SQ SN:chrUn_gl000215 LN:172545
@SQ SN:chrUn_gl000216 LN:172294
@SQ SN:chrUn_gl000217 LN:172149
@SQ SN:chrUn_gl000218 LN:161147
@SQ SN:chrUn_gl000219 LN:179198
@SQ SN:chrUn_gl000220 LN:161802
@SQ SN:chrUn_gl000221 LN:155397
@SQ SN:chrUn_gl000222 LN:186861
@SQ SN:chrUn_gl000223 LN:180455
@SQ SN:chrUn_gl000224 LN:179693
@SQ SN:chrUn_gl000225 LN:211173
@SQ SN:chrUn_gl000226 LN:15008
@SQ SN:chrUn_gl000227 LN:128374
@SQ SN:chrUn_gl000228 LN:129120
@SQ SN:chrUn_gl000229 LN:19913
@SQ SN:chrUn_gl000230 LN:43691
@SQ SN:chrUn_gl000231 LN:27386
@SQ SN:chrUn_gl000232 LN:40652
@SQ SN:chrUn_gl000233 LN:45941
@SQ SN:chrUn_gl000234 LN:40531
@SQ SN:chrUn_gl000235 LN:34474
@SQ SN:chrUn_gl000236 LN:41934
@SQ SN:chrUn_gl000237 LN:45867
@SQ SN:chrUn_gl000238 LN:39939
@SQ SN:chrUn_gl000239 LN:33824
@SQ SN:chrUn_gl000240 LN:41933
@SQ SN:chrUn_gl000241 LN:42152
@SQ SN:chrUn_gl000242 LN:43523
@SQ SN:chrUn_gl000243 LN:43341
@SQ SN:chrUn_gl000244 LN:39929
@SQ SN:chrUn_gl000245 LN:36651
@SQ SN:chrUn_gl000246 LN:38154
@SQ SN:chrUn_gl000247 LN:36422
@SQ SN:chrUn_gl000248 LN:39786
@SQ SN:chrUn_gl000249 LN:38502
@PG ID:bwa PN:bwa VN:0.7.15-r1140 CL:bwa/users/person/resources/reference/hg19/genome/ucsc.hg19.fasta 160095-T_S2_L004_R1_001.fastq.gz 160095-t_s2_l003_r2_001.fastq.gz
```

**BUT** when I use samtools to convert it to a bam file it's empty!

Can anyone advise?

My guess is that your paired files are not properly paired. The names of your reads don't seem to match between your pairs.

I get this error from read1 and 2 from lane 3 and 4 of this sample. all my other samples were fine. so I also thought something similar - so I tried all alignment combinations of read1 and 2 from the different lanes with the same problem - I also tried concatenation read1s from lane 3 and 4 and aligned this with read2s from lane 3 and 4.

is there a way to fix the different names of the reads?

How to fix it? Find the right pairs.

What does fastqc / multiqc look like? Any major flags? Is it possible that your forward / reverse reads are randomly sorted?

fastqc was fine - no flags. this is a problem with read 1 and read 2 from lane 3 and 4 of this sample. I have 29, and this is the only one with this problem