Subread and cutadapt working as Single-end reads BUT I have Paired end reads

[MohamedAbdalfatah]

Describe the bug
Hi, I have a Paired end reads and run the Analysis, it is working and the analysis finished but the output from the job looks like this:

[2023-09-18 20:22:30] started job on cnd22
This is cutadapt 3.7 with Python 3.11.0
Command line parameters: -a polyA=A{18} -j 8 -m 20 --nextseq-trim=20 --overlap 10 --json .//Sample_61_1/01.prep_map/Sample_61_1_cutadapt.json -o .//Sample_61_1/01.prep_map/Sample_61_1_ct2.FIFO .//C                                                NAG_61_1/01.prep_map/Sample_61_1_bc2.FIFO
Sep 18 20:22:55 ..... started STAR run
Sep 18 20:22:55 ..... loading genome
Processing reads on 8 cores in single-end mode ...
Sep 18 20:29:56 ..... started mapping
Finished in 13116.23 s (71 µs/read; 0.84 M reads/minute).

=== Summary ===

Total reads processed:             184,659,754
Reads with adapters:                18,918,659 (10.2%)

== Read fate breakdown ==
Reads that were too short:           2,847,809 (1.5%)
Reads written (passing filters):   181,811,945 (98.5%)

Total basepairs processed: 27,883,622,854 bp
Quality-trimmed:             444,255,550 bp (1.6%)
Total written (filtered):  26,351,946,868 bp (94.5%)

=== Adapter polyA ===

Sequence: AAAAAAAAAAAAAAAAAA; Type: regular 3'; Length: 18; Trimmed: 18918659 times

Minimum overlap: 10
No. of allowed errors:
1-9 bp: 0; 10-18 bp: 1

Bases preceding removed adapters:
  A: 0.0%
  C: 36.2%
  G: 28.0%
  T: 32.4%
  none/other: 3.3%

Overview of removed sequences
length  count   expect  max.err error counts
9       10      704.4   0       0 10
10      517717  176.1   1       177117 340600
11      304018  44.0    1       190575 113443
12      283249  11.0    1       184099 99150
13      262901  2.8     1       175160 87741
14      265188  0.7     1       176568 88620
15      259464  0.2     1       175629 83835
16      243957  0.0     1       166079 77878
17      308005  0.0     1       231961 76044
18      305998  0.0     1       230410 75588
19      254865  0.0     1       215364 39501
20      246792  0.0     1       210178 36614
21      244685  0.0     1       212938 31747
22      253005  0.0     1       223325 29680
23      231420  0.0     1       204443 26977
24      230055  0.0     1       205339 24716
25      228951  0.0     1       204991 23960
26      229941  0.0     1       207978 21963
27      233747  0.0     1       212649 21098
28      234700  0.0     1       214551 20149
29      220444  0.0     1       202363 18081
30      225177  0.0     1       207095 18082
31      211526  0.0     1       198189 13337
32      214492  0.0     1       200904 13588
33      208113  0.0     1       195351 12762
34      206794  0.0     1       194717 12077
35      209314  0.0     1       197655 11659
36      211980  0.0     1       201227 10753
37      205481  0.0     1       195499 9982
38      210830  0.0     1       200876 9954
39      209803  0.0     1       200376 9427
40      206090  0.0     1       196766 9324
41      205019  0.0     1       196402 8617
42      205028  0.0     1       196811 8217
43      202422  0.0     1       194839 7583
44      205056  0.0     1       198099 6957
45      194586  0.0     1       187225 7361
46      201689  0.0     1       194884 6805
47      201926  0.0     1       195093 6833
48      197043  0.0     1       190794 6249
49      196355  0.0     1       190106 6249
50      194592  0.0     1       188893 5699
51      191274  0.0     1       185619 5655
52      188547  0.0     1       183143 5404
53      188865  0.0     1       183705 5160
54      188712  0.0     1       183428 5284
55      181224  0.0     1       175992 5232
56      179210  0.0     1       174209 5001
57      180866  0.0     1       175753 5113
58      177193  0.0     1       172393 4800
59      170054  0.0     1       165498 4556
60      173613  0.0     1       168855 4758
61      165363  0.0     1       160839 4524
62      163250  0.0     1       158937 4313
63      161155  0.0     1       156876 4279
64      161480  0.0     1       157208 4272
65      161893  0.0     1       157896 3997
66      153574  0.0     1       149254 4320
67      151518  0.0     1       147728 3790
68      148780  0.0     1       145092 3688
69      148383  0.0     1       144982 3401
70      150129  0.0     1       146825 3304
71      147304  0.0     1       144014 3290
72      139829  0.0     1       136674 3155
73      136049  0.0     1       132698 3351
74      132149  0.0     1       128952 3197
75      136068  0.0     1       132830 3238
76      127073  0.0     1       124089 2984
77      121858  0.0     1       118660 3198
78      120923  0.0     1       117842 3081
79      122074  0.0     1       118690 3384
80      119286  0.0     1       116005 3281
81      114663  0.0     1       111655 3008
82      110632  0.0     1       107781 2851
83      110964  0.0     1       108004 2960
84      107549  0.0     1       104544 3005
85      107781  0.0     1       104811 2970
86      105902  0.0     1       102773 3129
87      103282  0.0     1       100426 2856
88      100748  0.0     1       97950 2798
89      100477  0.0     1       97567 2910
90      99183   0.0     1       96317 2866
91      99976   0.0     1       96824 3152
92      99037   0.0     1       96377 2660
93      92963   0.0     1       89691 3272
94      92750   0.0     1       89747 3003
95      91171   0.0     1       87992 3179
96      86348   0.0     1       83865 2483
97      83362   0.0     1       81012 2350
98      81222   0.0     1       78231 2991
99      83183   0.0     1       79989 3194
100     82015   0.0     1       79641 2374
101     78456   0.0     1       75905 2551
102     74701   0.0     1       72330 2371
103     77124   0.0     1       73998 3126
104     70341   0.0     1       67865 2476
105     70134   0.0     1       67729 2405
106     69577   0.0     1       66411 3166
107     65472   0.0     1       63165 2307
108     65461   0.0     1       63083 2378
109     62990   0.0     1       60878 2112
110     63160   0.0     1       60775 2385
111     63201   0.0     1       60654 2547
112     60084   0.0     1       58008 2076
113     59695   0.0     1       57883 1812
114     59491   0.0     1       57572 1919
115     61037   0.0     1       58644 2393
116     60751   0.0     1       58178 2573
117     60009   0.0     1       57255 2754
118     58885   0.0     1       56623 2262
119     57744   0.0     1       55523 2221
120     60774   0.0     1       58866 1908
121     45515   0.0     1       43567 1948
122     41845   0.0     1       39912 1933
123     40868   0.0     1       38948 1920
124     41013   0.0     1       38827 2186
125     40728   0.0     1       38730 1998
126     42023   0.0     1       39834 2189
127     41344   0.0     1       39021 2323
128     41129   0.0     1       38801 2328
129     41863   0.0     1       39405 2458
130     41025   0.0     1       38231 2794
131     39249   0.0     1       37094 2155
132     36322   0.0     1       34295 2027
133     35634   0.0     1       33543 2091
134     33615   0.0     1       31644 1971
135     30613   0.0     1       28625 1988
136     30484   0.0     1       28550 1934
137     30175   0.0     1       28143 2032
138     31347   0.0     1       29369 1978
139     33493   0.0     1       31190 2303
140     36774   0.0     1       34136 2638
141     37158   0.0     1       34209 2949
142     36100   0.0     1       33735 2365
143     33483   0.0     1       31149 2334
144     36191   0.0     1       34233 1958
145     41501   0.0     1       39033 2468
146     47419   0.0     1       44641 2778
147     54198   0.0     1       51620 2578
148     53832   0.0     1       50648 3184
149     43291   0.0     1       40350 2941
150     48441   0.0     1       45141 3300
151     109567  0.0     1       105750 3817
Sep 19 00:01:51 ..... finished successfully

        ==========     _____ _    _ ____  _____  ______          _____
        =====         / ____| |  | |  _ \|  __ \|  ____|   /\   |  __ \
          =====      | (___ | |  | | |_) | |__) | |__     /  \  | |  | |
            ====      \___ \| |  | |  _ <|  _  /|  __|   / /\ \ | |  | |
              ====    ____) | |__| | |_) | | \ \| |____ / ____ \| |__| |
        ==========   |_____/ \____/|____/|_|  \_\______/_/    \_\_____/
          v2.0.1

//========================== featureCounts setting ===========================\\
||                                                                            ||
||             Input files : 1 BAM file                                       ||
||                           o Sample_61_1_Aligned.out.bam                      ||
||                                                                            ||
||             Output file : Sample_61_1                                        ||
||                 Summary : Sample_61_1.summary                                ||
||              Annotation : Homo_sapiens.GRCh38.99.gtf (GTF)                 ||
||      Dir for temp files : .//Sample_61_1/02.featureCounts/tmp//exon          ||
||      Assignment details : <input_file>.featureCounts.bam                   ||
||                      (Note that files are saved to the output directory)   ||
||                                                                            ||
||                                                                            ||
||                 Threads : 8                                                ||
||                   Level : meta-feature level                               ||
||              Paired-end : no                                               ||
||      Multimapping reads : counted                                          ||
|| Multi-overlapping reads : not counted                                      ||
||   Min overlapping bases : 1                                                ||
||                                                                            ||
\\============================================================================//

//================================= Running ==================================\\
||                                                                            ||
|| Load annotation file Homo_sapiens.GRCh38.99.gtf ...                        ||
||    Features : 1376499                                                      ||
||    Meta-features : 60676                                                   ||
||    Chromosomes/contigs : 47                                                ||
||                                                                            ||
|| Process BAM file Sample_61_1_Aligned.out.bam...                              ||
||    Strand specific : stranded                                              ||
||    Single-end reads are included.                                          ||
||    Total alignments : 158035658                                            ||
||    Successfully assigned alignments : 141728594 (89.7%)                    ||
||    Running time : 19.95 minutes                                            ||
||                                                                            ||
|| Write the final count table.                                               ||
|| Write the read assignment summary.                                         ||
||                                                                            ||
|| Summary of counting results can be found in file ".//Sample_61_1/02.feature  ||
|| Counts/tmp//exon/Sample_61_1.summary"                                        ||
||                                                                            ||
\\============================================================================//


        ==========     _____ _    _ ____  _____  ______          _____
        =====         / ____| |  | |  _ \|  __ \|  ____|   /\   |  __ \
          =====      | (___ | |  | | |_) | |__) | |__     /  \  | |  | |
            ====      \___ \| |  | |  _ <|  _  /|  __|   / /\ \ | |  | |
              ====    ____) | |__| | |_) | | \ \| |____ / ____ \| |__| |
        ==========   |_____/ \____/|____/|_|  \_\______/_/    \_\_____/
          v2.0.1

//========================== featureCounts setting ===========================\\
||                                                                            ||
||             Input files : 1 BAM file                                       ||
||                           o Sample_61_1_Aligned.out.bam                      ||
||                                                                            ||
||             Output file : Sample_61_1                                        ||
||                 Summary : Sample_61_1.summary                                ||
||              Annotation : Homo_sapiens.GRCh38.99.gtf (GTF)                 ||
||      Dir for temp files : .//Sample_61_1/02.featureCounts/tmp//gene          ||
||      Assignment details : <input_file>.featureCounts.bam                   ||
||                      (Note that files are saved to the output directory)   ||
||                                                                            ||
||                                                                            ||
||                 Threads : 8                                                ||
||                   Level : meta-feature level                               ||
||              Paired-end : no                                               ||
||      Multimapping reads : counted                                          ||
|| Multi-overlapping reads : not counted                                      ||
||   Min overlapping bases : 1                                                ||
||                                                                            ||
\\============================================================================//

//================================= Running ==================================\\
||                                                                            ||
|| Load annotation file Homo_sapiens.GRCh38.99.gtf ...                        ||
||    Features : 60676                                                        ||
||    Meta-features : 60676                                                   ||
||    Chromosomes/contigs : 47                                                ||
||                                                                            ||
|| Process BAM file Sample_61_1_Aligned.out.bam...                              ||
||    Strand specific : stranded                                              ||
||    Single-end reads are included.                                          ||
||    Total alignments : 158035658                                            ||
||    Successfully assigned alignments : 142932586 (90.4%)                    ||
||    Running time : 17.85 minutes                                            ||
||                                                                            ||
|| Write the final count table.                                               ||
|| Write the read assignment summary.                                         ||
||                                                                            ||
|| Summary of counting results can be found in file ".//Sample_61_1/02.feature  ||
|| Counts/tmp//gene/Sample_61_1.summary"                                        ||
||                                                                            ||
\\============================================================================//

[bam_sort_core] merging from 72 files and 8 in-memory blocks...
[bam_sort_core] merging from 96 files and 1 in-memory blocks...
Number of distinct N supplied: 1147
Range of N: 1162
Number of features: 34981

As you can see Subread and cutadapt working as Single-end reads BUT I have Paired end reads, this is the FASTQ files I have:

Sample_61_1_S1_L001_R1_001.fastq.gz  Sample_61_1_S1_L002_R2_001.fastq.gz  Sample_61_1_S1_L004_R1_001.fastq.gz  Sample_61_1_S1_L005_R2_001.fastq.gz  Sample_61_1_S1_L007_R1_001.fastq.gz  Sample_61_1_S1_L008_R2_001.fastq.gz
Sample_61_1_S1_L001_R2_001.fastq.gz  Sample_61_1_S1_L003_R1_001.fastq.gz  Sample_61_1_S1_L004_R2_001.fastq.gz  Sample_61_1_S1_L006_R1_001.fastq.gz  Sample_61_1_S1_L007_R2_001.fastq.gz
Sample_61_1_S1_L002_R1_001.fastq.gz  Sample_61_1_S1_L003_R2_001.fastq.gz  Sample_61_1_S1_L005_R1_001.fastq.gz  Sample_61_1_S1_L006_R2_001.fastq.gz  Sample_61_1_S1_L008_R1_001.fastq.gz

This is the mapfile:

Sample_61_1    path/to/fastq     Sample_61_1

is this normal or what?
Best
Mohmed
Version
1.17.0

[zhouyiqi91]

It is normal. R1 reads are only used to extract barcode and UMI sequences. R2 reads were mapped to the genome and used for quantification.