map_element_barcodes (assign) [100%] 1 of 1, failed: 1 ✘

[Will19902225]

Hi,

I hope you are all doing well. I have an issue below. Could you please help me fix it? Thanks.

(MPRAflow) nextflow run --w /home/huanglabdell/Documents/MPRAflow/work02232024 association.nf --name HH_01092024as_1 --fastq-insert "/media/huanglabdell/596F-E992/02212024/BCL2/Data/Intensities/BaseCalls/HH_01092024as/3_S3_L001_R1_001.fastq.gz" --fastq-insertPE "/media/huanglabdell/596F-E992/02212024/BCL2/Data/Intensities/BaseCalls/HH_01092024as/3_S3_L001_R3_001.fastq.gz" --fastq-bc "/media/huanglabdell/596F-E992/02212024/BCL2/Data/Intensities/BaseCalls/HH_01092024as/3_S3_L001_R2_001.fastq.gz" --design "/media/huanglabdell/596F-E992/02212024/BCL2/Data/Intensities/BaseCalls/HH_01092024as/mIL13_hTNF_hIL6_hIL1B_remove_adaptor.fa" -dsl1
N E X T F L O W ~ version 22.04.2
Launching association.nf [chaotic_picasso] DSL1 - revision: 087b40d39f

                                      ,--./,-.
      ___     __   __   __   ___     /,-._.--~'
|\ | |__  __ /  ` /  \ |__) |__         }  {
| \| |       \__, \__/ |  \ |___     \`-._,-`-,
                                      `._,._,'

MPRAflow v2.3.1"

Pipeline Name : MPRAflow
Pipeline Version: 2.3.1
Fastq insert : /media/huanglabdell/596F-E992/02212024/BCL2/Data/Intensities/BaseCalls/HH_01092024as/3_S3_L001_R1_001.fastq.gz
fastq paired : /media/huanglabdell/596F-E992/02212024/BCL2/Data/Intensities/BaseCalls/HH_01092024as/3_S3_L001_R3_001.fastq.gz
Fastq barcode : /media/huanglabdell/596F-E992/02212024/BCL2/Data/Intensities/BaseCalls/HH_01092024as/3_S3_L001_R2_001.fastq.gz
design fasta : /media/huanglabdell/596F-E992/02212024/BCL2/Data/Intensities/BaseCalls/HH_01092024as/mIL13_hTNF_hIL6_hIL1B_remove_adaptor.fa
minimum BC cov : 3
map quality : 30
base quality : 30
cigar string : n
min % mapped : 0.5
Output dir : outs
Run name : HH_01092024as_1
Working dir : /home/huanglabdell/Documents/MPRAflow/work
Container Engine: null
Current home : /home/huanglabdell
Current user : huanglabdell
Current path : /home/huanglabdell/Documents/MPRAflow
base directory : /home/huanglabdell/Documents/MPRAflow
Script dir : /home/huanglabdell/Documents/MPRAflow
Config Profile : standard

executor > local (6)
[99/2602b6] process > count_bc_nolab (count) [100%] 1 of 1 ✔
[53/c27b7f] process > create_BWA_ref (make ref) [100%] 1 of 1 ✔
[3b/2d3496] process > PE_merge (merge) [100%] 1 of 1 ✔
[c7/e14a7f] process > align_BWA_PE (align) [100%] 1 of 1 ✔
[00/ec1417] process > collect_chunks [100%] 1 of 1 ✔
[02/caf11f] process > map_element_barcodes (assign) [ 0%] 0 of 1
[- ] process > filter_barcodes -
Error executing process > 'map_element_barcodes (assign)'

Caused by:
Process map_element_barcodes (assign) terminated with an error exit status (1)

Command executed:

echo "test assign inputs"
echo 30
echo 30
echo 3_S3_L001_R2_001.fastq.gz
zcat 3_S3_L001_R2_001.fastq.gz | head

echo count_fastq.txt
echo count_merged.txt
cat count_fastq.txt
cat count_merged.txt

python /home/huanglabdell/Documents/MPRAflow/src/nf_ori_map_barcodes.py /home/huanglabdell/Documents/MPRAflow 3_S3_L001_R2_001.fastq.gz count_fastq.txt s_merged.bam count_merged.txt HH_01092024as_1 30 30 n

Command exit status:
1

Command output:
test assign inputs
30
30
3_S3_L001_R2_001.fastq.gz
@M01416:220:000000000-DN9DG:1:1101:15719:1611 2:N:0:TTTCCTCT
TTTTTTTTTTTTTTT
+
111111>1000>EAE
@M01416:220:000000000-DN9DG:1:1101:17284:1628 2:N:0:TTTCCTCT
TTTTTCTTTTTTTTT
+
1>1>>1B3311>10A
@M01416:220:000000000-DN9DG:1:1101:15709:1633 2:N:0:TTTCCTCT
TTTTTTTTTTTTTTT
count_fastq.txt
count_merged.txt
1310484
0
/home/huanglabdell/Documents/MPRAflow
3_S3_L001_R2_001.fastq.gz
s_merged.bam
count_fastq.txt
count_merged.txt
counts
0
1310484
1310484
start
bad pairs: 0 poor quality: 0
start

Command error:

paired-end reads: 0it [00:00, ?it/s]
paired-end reads: 0it [00:00, ?it/s]

barcodes: 0%| | 0/327621.0 [00:00<?, ?it/s]
barcodes: 11%|███████████████▌ | 34592/327621.0 [00:00<00:00, 345912.91it/s]
barcodes: 21%|███████████████████████████████▌ | 70236/327621.0 [00:00<00:00, 349002.57it/s]
barcodes: 32%|███████████████████████████████████████████████▏ | 105901/327621.0 [00:00<00:00, 351260.75it/s]
barcodes: 43%|███████████████████████████████████████████████████████████████▍ | 142333/327621.0 [00:00<00:00, 355077.52it/s]
barcodes: 55%|███████████████████████████████████████████████████████████████████████████████▋ | 178835/327621.0 [00:00<00:00, 358000.26it/s]
barcodes: 66%|████████████████████████████████████████████████████████████████████████████████████████████████▏ | 215784/327621.0 [00:00<00:00, 361370.54it/s]
barcodes: 77%|████████████████████████████████████████████████████████████████████████████████████████████████████████████████▋ | 252876/327621.0 [00:00<00:00, 364181.15it/s]
barcodes: 89%|█████████████████████████████████████████████████████████████████████████████████████████████████████████████████████████████████▍ | 290323/327621.0 [00:00<00:00, 367206.76it/s][W::bgzf_read_block] EOF marker is absent. The input is probably truncated

executor > local (6)
[99/2602b6] process > count_bc_nolab (count) [100%] 1 of 1 ✔
[53/c27b7f] process > create_BWA_ref (make ref) [100%] 1 of 1 ✔
[3b/2d3496] process > PE_merge (merge) [100%] 1 of 1 ✔
[c7/e14a7f] process > align_BWA_PE (align) [100%] 1 of 1 ✔
[00/ec1417] process > collect_chunks [100%] 1 of 1 ✔
[02/caf11f] process > map_element_barcodes (assign) [100%] 1 of 1, failed: 1 ✘
[- ] process > filter_barcodes -
Error executing process > 'map_element_barcodes (assign)'

Caused by:
Process map_element_barcodes (assign) terminated with an error exit status (1)

Command executed:

echo "test assign inputs"
echo 30
echo 30
echo 3_S3_L001_R2_001.fastq.gz
zcat 3_S3_L001_R2_001.fastq.gz | head

echo count_fastq.txt
echo count_merged.txt
cat count_fastq.txt
cat count_merged.txt

python /home/huanglabdell/Documents/MPRAflow/src/nf_ori_map_barcodes.py /home/huanglabdell/Documents/MPRAflow 3_S3_L001_R2_001.fastq.gz count_fastq.txt s_merged.bam count_merged.txt HH_01092024as_1 30 30 n

Command exit status:
1

Command output:
test assign inputs
30
30
3_S3_L001_R2_001.fastq.gz
@M01416:220:000000000-DN9DG:1:1101:15719:1611 2:N:0:TTTCCTCT
TTTTTTTTTTTTTTT
+
111111>1000>EAE
@M01416:220:000000000-DN9DG:1:1101:17284:1628 2:N:0:TTTCCTCT
TTTTTCTTTTTTTTT
+
1>1>>1B3311>10A
@M01416:220:000000000-DN9DG:1:1101:15709:1633 2:N:0:TTTCCTCT
TTTTTTTTTTTTTTT
count_fastq.txt
count_merged.txt
1310484
0
/home/huanglabdell/Documents/MPRAflow
3_S3_L001_R2_001.fastq.gz
s_merged.bam
count_fastq.txt
count_merged.txt
counts
0
1310484
1310484
start
bad pairs: 0 poor quality: 0
start

Command error:

paired-end reads: 0it [00:00, ?it/s]
paired-end reads: 0it [00:00, ?it/s]

barcodes: 0%| | 0/327621.0 [00:00<?, ?it/s]
barcodes: 11%|███████████████▌ | 34592/327621.0 [00:00<00:00, 345912.91it/s]
barcodes: 21%|███████████████████████████████▌ | 70236/327621.0 [00:00<00:00, 349002.57it/s]
barcodes: 32%|███████████████████████████████████████████████▏ | 105901/327621.0 [00:00<00:00, 351260.75it/s]
barcodes: 43%|███████████████████████████████████████████████████████████████▍ | 142333/327621.0 [00:00<00:00, 355077.52it/s]
barcodes: 55%|███████████████████████████████████████████████████████████████████████████████▋ | 178835/327621.0 [00:00<00:00, 358000.26it/s]
barcodes: 66%|████████████████████████████████████████████████████████████████████████████████████████████████▏ | 215784/327621.0 [00:00<00:00, 361370.54it/s]
barcodes: 77%|████████████████████████████████████████████████████████████████████████████████████████████████████████████████▋ | 252876/327621.0 [00:00<00:00, 364181.15it/s]
barcodes: 89%|█████████████████████████████████████████████████████████████████████████████████████████████████████████████████████████████████▍ | 290323/327621.0 [00:00<00:00, 367206.76it/s][W::bgzf_read_block] EOF marker is absent. The input is probably truncated

barcodes: 100%|█████████████████████████████████████████████████████████████████████████████████████████████████████████████████████████████████████████████████▉| 327469/327621.0 [00:00<00:00, 368472.31it/s]
barcodes: 100%|██████████████████████████████████████████████████████████████████████████████████████████████████████████████████████████████████████████████████| 327621/327621.0 [00:00<00:00, 363754.83it/s]
Traceback (most recent call last):
File "/home/huanglabdell/Documents/MPRAflow/src/nf_ori_map_barcodes.py", line 157, in
save_barcodes_per_candidate(coords_to_barcodes, f'{prefix}_barcodes_per_candidate.feather')
File "/home/huanglabdell/Documents/MPRAflow/src/nf_ori_map_barcodes.py", line 134, in save_barcodes_per_candidate
pd.Series(d, name = 'n_barcodes')
File "/home/huanglabdell/Documents/MPRAflow/work/conda/mpraflow_py36-1978c54da7aacd41df3c7a4cb7639795/lib/python3.6/site-packages/pandas/core/generic.py", line 5063, in getattr
return object.getattribute(self, name)
File "/home/huanglabdell/Documents/MPRAflow/work/conda/mpraflow_py36-1978c54da7aacd41df3c7a4cb7639795/lib/python3.6/site-packages/pandas/core/accessor.py", line 171, in get
accessor_obj = self._accessor(obj)
File "/home/huanglabdell/Documents/MPRAflow/work/conda/mpraflow_py36-1978c54da7aacd41df3c7a4cb7639795/lib | 34592/327621.0 [00:00<00:00, 345912.91it/s]
barcodes: 21%|███████████████████████████████▌ | 70236/327621.0 [00:00<00:00, 349002.57it/s]
barcodes: 32%|███████████████████████████████████████████████▏ | 105901/327621.0 [00:00<00:00, 351260.75it/s]
barcodes: 43%|███████████████████████████████████████████████████████████████▍ | 142333/327621.0 [00:00<00:00, 355077.52it/s]
barcodes: 55%|███████████████████████████████████████████████████████████████████████████████▋ | 178835/327621.0 [00:00<00:00, 358000.26it/s]
barcodes: 66%|████████████████████████████████████████████████████████████████████████████████████████████████▏ | 215784/327621.0 [00:00<00:00, 361370.54it/s]
barcodes: 77%|████████████████████████████████████████████████████████████████████████████████████████████████████████████████▋ | 252876/327621.0 [00:00<00:00, 364181.15it/s]
barcodes: 89%|█████████████████████████████████████████████████████████████████████████████████████████████████████████████████████████████████▍ | 290323/327621.0 [00:00<00:00, 367206.76it/s][W::bgzf_read_block] EOF marker is absent. The input is probably truncated

barcodes: 100%|█████████████████████████████████████████████████████████████████████████████████████████████████████████████████████████████████████████████████▉| 327469/327621.0 [00:00<00:00, 368472.31it/s]
barcodes: 100%|██████████████████████████████████████████████████████████████████████████████████████████████████████████████████████████████████████████████████| 327621/327621.0 [00:00<00:00, 363754.83it/s]
Traceback (most recent call last):
File "/home/huanglabdell/Documents/MPRAflow/src/nf_ori_map_barcodes.py", line 157, in
save_barcodes_per_candidate(coords_to_barcodes, f'{prefix}_barcodes_per_candidate.feather')
File "/home/huanglabdell/Documents/MPRAflow/src/nf_ori_map_barcodes.py", line 134, in save_barcodes_per_candidate
pd.Series(d, name = 'n_barcodes')
File "/home/huanglabdell/Documents/MPRAflow/work/conda/mpraflow_py36-1978c54da7aacd41df3c7a4cb7639795/lib/python3.6/site-packages/pandas/core/generic.py", line 5063, in getattr
return object.getattribute(self, name)
File "/home/huanglabdell/Documents/MPRAflow/work/conda/mpraflow_py36-1978c54da7aacd41df3c7a4cb7639795/lib/python3.6/site-packages/pandas/core/accessor.py", line 171, in get
accessor_obj = self._accessor(obj)
File "/home/huanglabdell/Documents/MPRAflow/work/conda/mpraflow_py36-1978c54da7aacd41df3c7a4cb7639795/lib/python3.6/site-packages/pandas/core/strings.py", line 1796, in init
self._validate(data)
File "/home/huanglabdell/Documents/MPRAflow/work/conda/mpraflow_py36-1978c54da7aacd41df3c7a4cb7639795/lib/python3.6/site-packages/pandas/core/strings.py", line 1818, in validate
raise AttributeError("Can only use .str accessor with string "
AttributeError: Can only use .str accessor with string values, which use np.object dtype in pandas

Work dir:
/home/huanglabdell/Documents/MPRAflow/work/02/caf11fd327ea7f39e2b5ecfe90187f

Tip: when you have fixed the problem you can continue the execution adding the option -resume to the run command line

[visze]

Hi,

Looks like no PE reads could be merged in the step PE_merge because counting of the merged reads is zero. Can you give me an example from the forward and reverse reads (e.g. the first 10 reads)?

I recommend to make sure that the input of MPRAflow is correct. Also the command with some example files will help to understand you issue.

[Will19902225]

Hi, Thanks for your help. Here is a box link which you can download the raw reads data, R1, R2, R3. https://app.box.com/s/0l4tifwjzkdngtr96g82a3t030ihao41 Could you please take a look of these data? And if the data is not good, what can we do to fix it? Thanks!

…

________________________________ From: Max ***@***.***> Sent: Sunday, February 25, 2024 2:10 AM To: shendurelab/MPRAflow Cc: Gao, Junfeng; Author Subject: ⚠ EXTERNAL: Re: [shendurelab/MPRAflow] map_element_barcodes (assign) [100%] 1 of 1, failed: 1 ✘ (Issue #81) Hi, Looks like no PE reads could be merged in the step PE_merge because counting of the merged reads is zero. Can you give me an example from the forward and reverse reads (e.g. the first 10 reads)? I recommend to make sure that the input of MPRAflow is correct. Also the command with some example files will help to understand you issue. — Reply to this email directly, view it on GitHub<https://us-west-2.protection.sophos.com?d=github.com&u=aHR0cHM6Ly9naXRodWIuY29tL3NoZW5kdXJlbGFiL01QUkFmbG93L2lzc3Vlcy84MSNpc3N1ZWNvbW1lbnQtMTk2Mjg2NTU1Mg==&i=NWQ4ZWNjYmQ1OTJlNTkxNmZkNDVlNjZl&t=TjJIOFlrQUE4ZmRPcHdmcEY2S09hejBkaHZJcnU4WnlYV21TclZGV3BTdz0=&h=926fbe7a87eb4103a50afbc575ee49fd&s=AVNPUEhUT0NFTkNSWVBUSVZ-CzEl059Ym1RlqwKl3ZhNfC-dR2N5IDL9CUAdMA_6tA>, or unsubscribe<https://us-west-2.protection.sophos.com?d=github.com&u=aHR0cHM6Ly9naXRodWIuY29tL25vdGlmaWNhdGlvbnMvdW5zdWJzY3JpYmUtYXV0aC9BWkczRkEyNFdGVEE1SUZKUVVCNk02VFlWTDVZQkFWQ05GU002QUFBQUFCRFhJUEpaNlZISTJEU01WUVdJWDNMTVY0M09TTFRPTjJXS1EzUE5WV1dLM1RVSE1ZVFNOUlNIQTNES05KVkdJ&i=NWQ4ZWNjYmQ1OTJlNTkxNmZkNDVlNjZl&t=aXplV3NBRU9hSWUrcUxrWDA1d3pnM1NHb2tScEIyUTlVanlHcEREa0V1MD0=&h=926fbe7a87eb4103a50afbc575ee49fd&s=AVNPUEhUT0NFTkNSWVBUSVZ-CzEl059Ym1RlqwKl3ZhNfC-dR2N5IDL9CUAdMA_6tA>. You are receiving this because you authored the thread.Message ID: ***@***.***> CAUTION: This email originated from outside of the organization. Do not click links or open attachments unless you recognize the sender and know the content is safe.

[Will19902225]

Hi,

Looks like no PE reads could be merged in the step PE_merge because counting of the merged reads is zero. Can you give me an example from the forward and reverse reads (e.g. the first 10 reads)?

I recommend to make sure that the input of MPRAflow is correct. Also the command with some example files will help to understand you issue.

Hi,

The R1 reads first 10 reads
@M01416:220:000000000-DN9DG:1:1101:17453:1773 1:N:0:TAGATCGC
TGGTTGCAAGGGACCGTCGACCTGAGGAGATCGGAAGAGCACACGTCTGATCTCCAGTCTCTTCTTGTTTTTTTTTTTTTTTTTCTTTTCTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTCTTTTTTTTTTTTTTTTGTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTCTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTGTTTTTTTTTTTT
+
1>A>AAC1BFAAACAEAAEEAEEHG0FFCCGHFC?/ECFFFHFHGHHGE12DFF111D111221D111100B/>/>//>/<///0?1112??111//>A-----;---99--9-;------9-------9///9//--------;------99@@@?;---9-9--;-9--------;--@--=//9///------------;-@-9@@@--@-@---99-@?=@-99--9-=@@-@@
@M01416:220:000000000-DN9DG:1:1101:12921:1774 1:N:0:TAGATCGC
TCGAAATGAGGGAGGTTGGTCGCCGCAGATCGGAAGAAGCACACGTCTGATCTCCAGTCACCACTATTGTGTTTTTTTTTTTTGCTCTTCTTTCTTCTTTTCTTTTTTTTTTTTTTCTTTTTTTTTTCTTCTCTTTTTTTTTTTTTTTTTTTTTTTTTTCTTTTCTTTTTTTTTTTTTTTTTTTCTTTTTCTTTTTTTTTTTTTTTTTTTCTTTTTTTTTTTTTTGTTTTTTTTTTTT
+
3>A?AAAFFDB?2FC2FGEEEEEEEEECEGHHGGGCAGHHHHFFCGHFF55BF5FF3D33533?B3555B4?3B2B//>/////1B11112B12212BB11211?11/-A-/0000;-----/0;000;00;0------;--------;--9--////////99/------9@-=B-9-////////9///------9-9--9-9//B/F/---@-@B9B-9;FB:;-9-9??
@M01416:220:000000000-DN9DG:1:1101:14672:1775 1:N:0:TTGATCGC
TGAGATAGATACAATCAGCCACTAGCTTGTACATGTGCACCACACACACACACACACACACTCACTTTTTTGTTTTTTTTTATTTCTTTTTTTTTTTTTTCTCTTTTTTTTTTTTATTTTTTTCTTTTTTTCTTTTTTTTCTTTCTTTTTCTTTTCTTTTTTTTTTTTTTTTTTTGTTTTTTTTTTTTTTTTTTTTTTTTTCTTTTTTTTTTTCTTTTTTTTTTTTGTTCTTTTTTCT
+
3>>>?FFFFFFFGBGFBGG4FFFG44FGBHGHFFBGHGGH3EGH?EGEAA22E21EE111133335535A0022?21////144B4B4443>////>///011?11<1@-<-@-;.0<000--/;0000;@/0000;@9-///;////;//:/;////9/9-------9---;-9-...9.--9-----;-9--9---@--/////;9-----///;/:@>------:.//;///;//
@M01416:220:000000000-DN9DG:1:1101:14624:1784 1:N:0:TTGATCGC
TGTGCCTGCGTCACCTCTGACCACACAGCAGCCCAGACAAAGCAGGGCTGGGGGTGGCTTTTGACTCTGCTTTTTTTTTTTCTTTTTAGTTTTCTTCTTTTCTTTTTTTTTTTCTTGTTTTTTTTTTTTGTTTGCTTGTTTTTTTTCTTTTCTTTCTGTTTTTTTTTTTTTTTTTCTTTTTTTTTTTTTTTTTCTTTTTTTTTTTTTGTTTTCTTTTTTTTTTTCCTTTGTTTTTTTT
+
1>>A1BFFFDADGC1FGGGGCGGAGEAHGHHHGFEFFFFFFFBHHGE?EEGG/EGCC0B0B11/111111B1@11///>/<01B111/0B22221B2B1112?111<>//---/=0000//<-A-A--A-////0;00.09B.---/;9/////;////;/-;-9------9@--////;/--;-9-------/;////-----;---9;--////9/;----9///;//;-------
@M01416:220:000000000-DN9DG:1:1101:14078:1785 1:N:0:TTGATCGC
TGTGCCTGCGTCACCTCTGACCACACAGCAGCCCAGACAGAGCAGGGCTGGGGGAGTTTCTTTGTCCCTTTTCTTTTTTTTCACTTTGGTTTTTTTTTTCTTTTTTTTTTTTTTTTTTCTTTTCTTTTTTTCTCTCTTCTTTTTTTCTGTTTTTTCTTTTTTTTTTTTTTTTTTTTTTTTTCTTTTTTTTTTTTTTTTTTTTTCTTTTTATTTTTTTTTTTTTTTTTTTTTTTTTCTT
+
3AAABBFF4DADC?GFGGGGGCCG4EEFHHFH2EEAGGCCGGFFHHCGHGDDEE0>?BF5BF55B553B3B3444443//E34444B33//0////B//0?1?1<//-----<-;---/000;000000--//////;/;////;-///;//..-/;99//--;-9-@--@9-----;---/;9///--;>----99----;-/;/://./;/;/---9-9@-9-;----9----/;/
@M01416:220:000000000-DN9DG:1:1101:13957:1791 1:N:0:TTGATCGC
TGAGATAGATACAATCAGCCACTAGCTTGTACATGTGCACCACACACACACACACACACACTCACTCTCTCTTTTTTTTTTGTGTTCACTTCTGTTTTCTTGTTTTTTTTTTTGTTTTTATTTTGTTTTCTTTCCTTTCTGTTTTTTTCTTTTTTTCTTTTCTTTTTTTTTTTTTTTTTTTTTTTTTTCTTTTTTTTTTTTTTTTTTTTCTTGTTGTTTTTTTTGTTTTTTTTTTTTT
+
3>3AAFFFFFDFGGGFGCGGGGHHHHHGHHHHHHGHHGHHFHHCDEGGGFDEGGGFG111A33335553333555B0>////11?344?4?3B4?440?443B300/><>/@//?0?0?/?11<1<0<01<1111>>11110<=/.--/<0000;-/;00000009;------99-;-99-9-----;/9////--;>-B---;;BB--////;/.9.9..9@B-;9...9;9----9
@M01416:220:000000000-DN9DG:1:1101:15225:1791 1:N:0:TTGATCGC
TGAGATAGATACAATCAGCCACTAGCTTGTACATGTGCACCACACACACACACACACTCTCTCTTTTTCTTTTTTTTTTTTTTCTGTTCTTTTTTCTTCTCTTGTTTTTTTTTTATGTTTTTTTTTTTTCCTTCTTTTTTTTTTTTTACTTTTCTTTTTTTTTTTTTTTCTTTTTTTTTTTTTTTTTTTTTCTTTTTCTTTTTTTTTGTCTTTTTTTTTTTTTTTTTTTTTTTTTTTT
+
1>>AAFBFB331A1F1A11111A1GB3D0BE3GBGDBBHCC0A0EFCFG/ABE/EA/00111A1211B011111>////>//>0B2222B1111>011221>21>100?/</<-.111110.<----:;/00000C000--9----9-/;/;//////;-9-----;-9/;////----9-9--9---;-9/;/9:///////9-9--9/9//9B--9-9>--9-------------;
@M01416:220:000000000-DN9DG:1:1101:16193:1792 1:N:0:TTGATCGC
TGGTGGCTCATATTAGTCGGTGTATTAGATCGGAAGAGCACACGTCTGAACTCCAGTCACCTCTAGTGGGTATTTTTTTTTAGCTCGACTTCCTTCTTGTCATTCCGTTTCTGTGCAATGTTTTCTCTTCGCTATGCGTTTGCTATGTATCTCCTTCTTGTTTCCGTTCGCCTCCATTTTGATCAAGTTCGTGTTGCCGTTTCTTTTCTCTTTTTCCGTCTTCTTCTTGTTTTTTTTT
+
3ABABA?FFFFDGFDDFGGGCCAGHHHHHHGEGCGDHFGGEHHGHGHGHGHFHHHHHE3BF3BF355511B5B5F55>0//133BB1/>?E43343BF33?44B4433B33BBF434343B2202122BB//C//11/////1?111?<11111?F?111>1011.1><.-..../=00=0.00000000:.....00-..//;00C0C00;0;0/0.;.CE0B00CF000/9.--9=
@M01416:220:000000000-DN9DG:1:1101:15141:1793 1:N:0:TAGATCGC
TAAACAAACAAACCACTCTTGACTCGACCTCTTGACGGGAAGGTGGCTATGGACACATCTCTTGTCTTTTTTTTTTTTTTTTCTTTTTTTTTTTTTTTTCTTTTTTTTTTTTTTTATTTTTTTTTTTTTTTTTTTTTTTTCTTTTTGTTTCTTTTCTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT
+
1>>>AF1C1BA1AA11FCEEGGHHHGE?0B1AFHBGG?CCAAC11/0CDFDF11BA011D1D211122AD1/>///>E////01>1B1/</</---<--/000;0-:?A?9?@-@-/;//9---9-99-;;-----;;9-///9//9-;-/;;B//////;-9--9--;-9-9------9---;-9------9---99--9-----9@----9---99-;@@;---@-@----9----
@M01416:220:000000000-DN9DG:1:1101:17575:1798 1:N:0:TTGATCGC
TCGAATTACGTGGGCGGTAGAGTATAAGATCGGAAGAGCACACGTCTGAACTCCAGTCACCACTAGTTGGTTTTTTTTTTTAGCTCTACTTTTTTCTTTTCTTTTTTTTTTTGTTCTTTGTTTTTTTTTCGCTTTTTTTTTTCTTTGTTTCTTTGGCTTTTTTCCTTTCTCTTTTTTTTTTTTTTTTTTCTTTGTGCCTTTTTTTTTTTCTTTTTCTTTTTTTTTCTTTTTTTTTTTT
+

3AAACB5FAFFGGGG?AEEFGHFHFHHGFDF?2FGGHHGHHHGHHHGHFCF5BA3D33D331B?35553B1B1F//>////?3B443BBF211/0BB11211?11//>-->-</00000=///.---;/-.;..00----9/;///;FF///;////.//;-///9;////////;--9---------//////.///////9-99-B//;/B////;/;@-;-9BF///--;-;=>
@M01416:220:000000000-DN9DG:1:1101:14363:1798 1:N:0:TTGATCGC
TAAGTTGGGGACACACAAGCATCAAGGATACCCCTCACACTCCCCATCCTCCCTGCTCCGTTTCCTATTTTGTTTTTTTTTGCCTCTGTTTTTTTTTTGTTTTTTTTTTTTTATTCTTTTTTCCGTCCTCTTTCCTCTCTTTTTTTTTTTCTTCCTTTTCTTTTTTTTTTTTTTTCTTTTTTTTTTTTTTTTTTTTCTTCTTTTTTTTTTTTTTTCTTTTTTCTTTTTTTTTTTTTTT

1>>AAFF1>AADAF1EEFFCGGHHHBCH11GHF0AAFC1BBBGAEFGFHHGG/A1AF11//BAA212212220>0BF/>///1111B122B0/>EE/>/B0?0////-->-<.<00=000:-/0.<...00;00000;/00000-;-;@-//;///;9F/;/BFB-;;------9/;/BF/@---;@@--;9-;--/:B////9/-@----;-9-///9/99/;9///----@-----
@M01416:220:000000000-DN9DG:1:1101:12672:1800 1:N:0:TTGATCGC
TAACTCCAGTCACCACTAGAGGGTATATAATGGAAGCTCGACTTCCAGCTTGGCAATCCGGTACTGTGCAATGTTTTTTTTTCTCTTTTCTTTTTTTTATTTTCTTTTTTTTCTGTCTGTTCTTCTTCATGTTTTGTTTTTGTTTTGTGCCTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT
+

3>AAF4FCD4FFFGGF4F4GFCEGHFHGHFFDGBGCFGEEGEGHHF5FGHGEEF3F3210AEAFBF55555AD55210//>344B444444443/>//44?4444443///01222211@2211?111@11?1000000.-00000001100000<--;-;--;-@=---;-;@-;--;B-9999---=9>=?=?>@b?-;9>BBB>BBB??B;>=??@bb?=-;9;@?>;?BB?;@
@M01416:220:000000000-DN9DG:1:1101:15759:1815 1:N:0:TTGATCGC
GTCGGAAACGGCGGCTCGGCGCGCAAAGATCGGAAGAGCACACGTCTGAACTCCAGTCACCTCTTGTGGGTTTTTTTTTTTAGCTCGACTTCTTTCTTTGCAATTTTTTTTTGTGCAATGTTTTTTCTTCGCTATGCTTTAGTTTTGTACCGCATTGTTCGTTTCGGTTGTCTCCTTTGTGTTTTTGGGTTTGGTTCCTTGTTTGTTTTCGTTTTCCTTTTTCTTTTTTTTTTTATTT

1>>AAD1DFA1@CEGECEEGGAEC/EEF0FGFE/BEGCFG0FFEFHHGBB1GFHHHH11FF1BG1111/<?/?/<//>//>?<1?1<-><C111=1>G00000=0000<--:-9/00000;00/.-/0;0..;..;0000///;//-;///----9/;//-;/-/--;---/////;//;-9/-----------9-///////-/9;--/-;/;-///;/BB/9;/9;-------///
@M01416:220:000000000-DN9DG:1:1101:14344:1820 1:N:0:TTGATCGC

There are such instances in the remaining reads in R1
@M01416:220:000000000-DN9DG:1:1101:13255:1910 1:N:0:TAGATCGC
CAAACAAACAAGCCTAAAATTTCCCTTTAGATCGGAAGAGCACACGTCTGAACTCCAGTCACCACTAGAGGGTATATAATGGAAGCTCGACTTCCAGCTTGGCATTCCTTTACTGTGCAAAGTGAATACATCGCTAAGCGAAAGCTAAGTGTAGACGCGCTTAGACCGGTCGCCACCATGGTGTGCAAGGGCGAGGAGCCGAGGCTGATCTCGTATGCCGTCTTCTGCTTGTTAAAAA
+
BBBABFB??CAFFFF4FGDGGGHHHHHHHHHHFAGEGGHFHHCHHEGHHFHHHHGHHHHDGHFHAGHHFGFGE335D55D5FGHHGHHC//EFEB3FGHH3EHH3B4@44B3BEEGHHFHD3FB444BBDGBEEHG/3EB?//<0BDGHHHHH12DD@CCGGDB<G<<@C@-<<....:0/C:.;:0CCE.E---9-A.99-A?=EF.9//;.A.9///-ABFE;FFFFFF.//////
@M01416:220:000000000-DN9DG:1:1101:14322:1910 1:N:0:TTGATCGC
GACATACGAACCACTCGCTGATGCCGAGATCGGAAGAGCACACGTCTGAACTCCAGTCACCACTAGAGGGTATATATTGTAAGCTCGACTTCCAGCTTGGCAATCCTGTTCTGTGCAAAGTGAACACATCGCTAAGCGAAAGCTAAGCCAATCAATTTGGAAACCGGTCGCCACCATGGTGAGCAAGGGCGAGGAGCCGAGGCTGATCTCGTATGCCGTCTTCTGCTTGAAAAAAAAA
+

AABAFFFAADBGGGCFGGGGGHBGGGFEFHFEECG2GCHGHHGHFHGHEFHFHHHHHFHHHGHHHDFGEEHHDF44B444@FFEHGG?EG4FGFFHHHGHHHH3243B3BBFFHHHHHHHHBGFFHEHH?EGGDHECD/B/<<F1CGHGGHHHHHHHFGEFF0FD---ACFGGCGFFBGHB./0CGEDAG?D--9C9B?DAFF..9/;BAEDFFFF-ADFFFFFFEFEF/9/99-9-
@M01416:220:000000000-DN9DG:1:1101:12058:1914 1:N:0:TAGATCGC
GGATCTATCAGTTTGCCTCCTTCAGCAATCGGAAGAGCACACGTCTGAATTCGAGTCACCACTAGAGGGTATATATTTTTTGCTCGACTTCCATCTTGGCTATCTGTTTCTGTGCAAAGTTTACACTTCGCTAAGCGAATGCTAATTTAAAATTAAATCTTTCCGGTCGCCACCATTTTTTGCAAGTTCGTGGAGCCTTTGCTGATTTCGTATCTTTTCTTCTGTTAGAAATTTTTTA

A@BBAFFFD55DGFFBGEE4GDFGGFGFGFEEG22B22B2BAEEGF25F5DG2BBFGFF3BABC333G1AFEBD55F5550>3FE1>EEE4334BFGF3B33BF444B44B4@B?4?3BB44B4433BBFDE//33<//11F221?2@211F1>122>22@1//AC/<...<.>11>1--00=0000<.....:C/000;00000<9F.//.000;0;C0;00=0000000000-.
@M01416:220:000000000-DN9DG:1:1101:16586:1915 1:N:0:TAGATCGC
CTGGAAGTTAGAAGGAAACAGACCACAGACCTGGTCCCCAAAAGAAATGGAGGCAATAGGTTTTGAGGGGCATTGTGTCGGGGTTCAGCTTCCAGTGTCCTACACACATATCAGTCAGTGTCCCAGAAGACCCCCCTCGGAATCGGAGCAGGGAGGCTGGGGAGTGTGAGGGGTATCCTTGATGCTTGTGTTTCCCCAACTTTAGATCGGAAGAGCACACGTCTGAACTCCAGTCACC
+

?AA>CCCFFFF5GGA44BGFGCA4ECG4FDCHFEDB2FF2BGF223FGF32AEF1FFBFHFHGF1AGGGCCC33@5251/>?EEHFHH4B3@3B43B44GHHG03//33B4BFBFG?4F?4F?3F/B?BFFG?@/BA<AA/F1??/<CHHH?.A-.<.<.-.:CCFC0G..C::G00CF0BF0000BFFF//900999.CFB9CBB99.A?D..9;.B.-;.B;///;/9B/;BBB/
@M01416:220:000000000-DN9DG:1:1101:15876:1919 1:N:0:TAGATCGC
GCCTAGAGCTGTGTCCATAGCCACCTGTCAAGAGGTCGAGTCAAGTCACTGTGAAAACTAAAAGTCCACAGTGCCATCTCGAGTGGAAAGTTATCCCCTCGCAGTGTGTCTTAGAAACTCCCCCAGCCCTGCTCTGTCTGGGCTGCTGTGTGGTCAGAGGTGACGCAGGCACCAGATCGGAAGAGCACACGTCTGAACTCCAGTCACCACTAGAGGGTATATAATGGAAGCTCGACTT

AABBBFFF5BC5FGCGGGGFGFFFGFGHHHBB4FGHHEGGHHHHF5GBF5G5DG5FBEBG3F3GAFBGFGG3F5B5F3FACGEG1AGHE1D5@F5@GFFGCEEE13B31BF4B@G4FG3?FG/EC/AAEGHHFBEFHHFGBHAG//FG1BGHD0DD0D1/>//?BGEFGCE?E4B444BF3EFGBB3444B44BBF?E4B?GF12BBB0/BFD?F?BF0B///?0<11?0?GG--<>==GF11A...<AA11BCD3FDF1GCFGGGFBF000G0BEFGHGHFHFF0EA0B/G1DGHFG?A/BG1FFF1FFH/FBE?GC0111112///>>/GEB10B0/010>0BGF>CC01/01BB12>2>1FGF>D111B11FGHH/?>/>A@///?<///>@CGGCECG.CG.<..@C.CFCG..:C.G000<90;09B0/;/EFFF0;./..;BF0CBB9..9-9---/9--///::9//;;//9/:/99
@M01416:220:000000000-DN9DG:1:1101:14831:1921 1:N:0:TAGATCGC
GAGACCACCAAGGCAAGCAAGAGCCTGAACTCCAGTCACCACTAGAGGGTATATAATGGAAGCTCGACTTCCAGCTTGGCAATCCGGTACTGTGCAAAGTGAACACATCGCTAAGCGAAAGCTAAGACAACAGTAGAAAATACCGGTCGCCACCATGGTGAGCAAGGGCGAGGAGCCGAGGCTGATCTCGTATGCCGTCTTCTGCTTGAAAAAAAAAACTTTTTATTTGTACTTTTCT
+
BBBBCCFBCCAFGGGGCGGGGFHHHCGHHHHHHHGHHHHGHHHHHDFGGAFGHFFHHHHHHHHHHGGGGGHHHB3FH3133BCGHGGE@>G4FFBEHBFGHFHB421B?//FEEGHGFEFGFHGHHEGHHHHH3FFHEGFHFHFG?CCGGFGGHHHGHDG1GFHFGGDFGGGGCGGFGC-??EGCF0CBAAEFG0:@ECFGGGGEFFF.09F.--;--.00000.00000000:000:
@M01416:220:000000000-DN9DG:1:1101:15375:1922 1:N:0:TTGATCGC
GGAGATAGATACAATCAGCCACTAGCTTGTACATGTGCACCACACACACACACACACACACACAAGATCGGAAGAGCATTCGTCTGAACTCCAGTCACCACTAGTTTGTTTATAATGGAAGCTCGACTTCCAGCTTGGCAATCCGTTACTGCGCAAAGTGAACACATCGCTAAGCGAAAGCTATGTATTGACATTAATCAACCGGTCTCCACCATGTTGAGCATGGGCGAGGAGCTGA
+
ABAA?FFFFFFFGC5FGCG4GGGF44DFGHE5B6FGCHDFFCGF?EE?FAEEGGCED1BA?EAF1AFGF1>B1111B3355B3B113434B333B3@3EDEG3ED444B0B44B?4F3BDC/3F<//?/B?GDFGB0/0?1?10?/1<<2>1?<<@@0??DDGG0FG1.<CD.0/-.-<.C000<;0G:00C:CCG0C0;::G---9.0;C/F.090900C0009C.9--9999..9/
@M01416:220:000000000-DN9DG:1:1101:19463:1922 1:N:0:TAGATCGC
GGTGCCTGCGTCACCTCTGACCACACAGCAAGTTCGTTAGAAATGAATGAAGATAATGAGGCCTCGTGACTTTTATTTTTCACAGTGAGTGGGTTCACTGTGGGTTTTTTTGGCTATGTATACTGCTCTAGGCAGATCGGATGAGCACACGTCTGAACTCCAGTCACCACTAGTGGGTATATAATGGAAGCTCGATTTCCAGCTTTGCAATCCGGTACTTTGCTAAGTGAACACATCG
+
AAABACFB?DAA?EGFGGB5BAGFHC?GDGBGHFAFGBAFD55FG55FAA5BEFFFHBD531AFA?GHFFGHEA55DGGH3DDFFHHEF531?111@B4BF4BEE1111/>//?F3FGHH4444B3B3F3?222<FF2C/A//222<02?A0/?<GGBF1?<GGG111>11<.<0<<==00D0<DG.0C.-/;:00;0:CC0::0<;:/.9D-.00;F00000;BBFB0/.;0.
@M01416:220:000000000-DN9DG:1:1101:19606:1922 1:N:0:TAGATCGC
GCCTAGAGCTGTGTCCATAGCCACCTTCCCCCACAAACTCCCCCAGCCCTGCTCTGTCTGGGCTGCTGTGTGGTCTTTGTTGACGCAGGCACCTGATCGGAAGATCACACGTCTGAACTCCAGTCACCACTATAGGGTCAATCCGTTACTGTGCAAAGTGAACACATCGCTAAGCGAAATCTAAGTTCCTATCATAATCCACCGGTCGCCACCATGTTGAGCATGGGCGAGGAGCCGA
+

AAAAFFCFFFFGFCGCBGGGGBGAGFHHBHCEEGGF2FFGEEEAEFGEEACFHHFGGHH32CFGAHFH3FEEG355555B?BDEF/EE1?1?3B33BE11?E0??3333/B?GF3?GFG3B3BB?32BEFH3BBD0FF02>>2?//FFFGF1@FGBDGB11F0><..>A<10-.--.<==0=DGD0G0;:0CH00:0C/.::AB--9.9..;00000/0009...-=A--;.9--
@M01416:220:000000000-DN9DG:1:1101:16661:1923 1:N:0:TAGATCGC
GCCTAGAGCTGTGTCCATAGCCACCTTCCCCCACAGTGCCCCCTCTCACTGTGAAAACTAAAAGTCACGAGGCCTTATTTTCTTCATTCATTTCTAACGAACTTTCCTCTTTCAACTGATTTCTCGTCAGCCAGGGAGTGCCCCATGACGAAGCCACTCGAGTCAAGGGGTCGAATTTTCTACTTATGACCTCACTTTCTGTTGCTCTTTCTTTCCCTCCCTCCACAGTGCCTTCTCT

3AAAAFFFFFFFFGGFGGGGC4FFCGHHG4FEEEAGFHHGFFEA2FG3FGFHFGGHBCG5BBBFBDF5E1A0E1@33DF5BGEF55@4F@B4FDFE4FAC?/GDF434343B44GHBG3DGG444/?//?BGHEHG/?/B1B0//B?<FD/@/<00F?0?/<F01?11<-<AC-.CCGH0==0DG0<00=<D0/.<CH0CGGG0;C0;0<CC;00;00/9.9/./9/.;0;00;0900
@M01416:220:000000000-DN9DG:1:1101:13793:1927 1:N:0:TAGATCGC
GGAGATAGATACAATCAGCCACTAGCTTGTACATGTGCACCACACACACACACACACACACCAGATCGGAAGATCACACGTCTGAACTCCAGTCACCACTAGTGTTTATATTATGGAAGCTCGACTTCCAGCTTGGCATTCCGTTACTGTGCAAAGTGAACACATCTCTAAGCGAATTCTAATACCCTCCAAACACTAACCGGTCGCCACCATGGTGATCTTTGGCGAGGAGCCTAGG
+
AA3ABFFFBFDF5EGEFFFBGFHFGHGHBHFGGHFHFFFBGHHH2EFEGGCD2E11A111B1A11AF1E1F?15533330BBFFF4CGG3@3B333B@GFCE4BB@G4B4?4?BDG33FGGC/>/?EDFDFFH10/011B?F?1@?FHHHGFFB10@11F1F0/?11FFGB1<-.--.<<<111<0CGH?CG0<.<0<<G.--=:-@e....0C/000000009/C-;;9B?.;/9//
@M01416:220:000000000-DN9DG:1:1101:14877:1929 1:N:0:TAGATCGC
AAAGTCCCAAACCAACTGCTGGCCTGCCCAAGAAAGAAACCAAATTCATACAACCTCCGCAACTGAGATTGAAACCATGATAGATCGGAATCGCACACGTCTGAACTCTAGTCACCACTAGAGGGTCTATAATGGAAGCTCGATTCCAGCTTAGCAATCCGGAACTGTGCAAAGTGAACACATCGCTAAGCGACAGCTAAGAAAACTCTACTAATCAGTGTTCTCCACTATGGTGAGC
+
AA?AADF1DCC?111A1BGGGCGHCG00A1A00ABEGH000BAGF1FD2FGFFG/FFGC/AE?EGF11B11F111BF1D1DF@FGHEC?//10//>>0EGHB/F21F1111B121BGCC01B10/?00>22BF21F1GCFG//0212@101@11@FGGCGE@//>11>11>1><1111<10<C0..<.<<C-.--;.C00:00/:/;F009CB00900090;C009090;C000;F?/
@M01416:220:000000000-DN9DG:1:1101:17264:1930 1:N:0:TAGATCGC
GGGTCTCACAGGGTCACGTCCGCTCAAGATCGGAAGAGCACACGTCTGAACTCCAGTCACCACTAGAGGGTATATAATGGAAGCTCGACTTCCAGCTTGGCAATCCGGTACTGTGCAAAGTGAACACATCGCTAAGCGAAAGCTAAGCGCTTGAACAATCCTACCGGTCGCCACCATGGTGAGCAAGGGCGAGGAGCCGAGTCTGATCTCGTATGCCGTCTTCTGCTTGAAAAAAAAG
+
A?AAAFDDFFBA2B4FFECFC?EEGDFGFHHHFGGGHCHGHCFGHHHGHFHHHHHHHHHGHBHHHGGHFDEFHFG55DD533BGGHGGGGGHE3FBGHGGFHFH?0//>><GFGDFHHHHHEHFG1BGGHGGHGFHHCEC//0GF1GFG/FFAHCGFGHGFHGG0<-@d?CEG?.GHHFGHGF/CG/EDG@@g@9E..-;@..;BB//9FFFFFF9/@A=FFFFFFFF/.;/;9@--.
@M01416:220:000000000-DN9DG:1:1101:13847:1931 1:N:0:TAGATCGC
GGTGCCTGCGTCACCTCTGACCACACAGCAGCCCAGACAGAGCAGGGCTGGGGGAGTTTCTTAGGCCCTCTGCGCGGGGTTCACTTTCCACTCGAGATGGCACTGTGGAGGGGCAGATCGGAAGAGCACACGTCTGAACTCCAGTCACCTAGAGGGTATATAATGGAAGCTCGACTTCCAGCTTGGCAATCCGGTACTGTGCAAAATGAACACATCGCTATGCGAATGCTAAGAACTG

The R3 reads, first 10 reads
@M01416:220:000000000-DN9DG:1:1101:17453:1773 3:N:0:TAGATCGC
TTTCTTGTCGTTTTTTCTTTTCTTCTTTTTTCTTTTGTTCGTCTTTTTTTTTTTTTTTTTTTTGTTTTTCTTGTTCTTTCTCTTTTTGTTTTNCTTNTTTTNTTTNTTTNNNNTNTNTTTTTTTNNNNTTNTTNNTNTTTNAATANANNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNGGNNGNNNNTNNTNNNNNNNNNGNTTCTCTTGTTGNNNNNN
+
11>1131B11>111A001AB131333B311A011B11001/B//ADB1/////////>/>//>/0000?0>111112>1222?2111/<00?#//?#/>..#.<>#<.<####<#<#=<.=.--####..#.9##9#;9.#9...#9##################################################-9##-####-##9#########-#-:-;-9;/9/-######
@M01416:220:000000000-DN9DG:1:1101:12921:1774 3:N:0:TAGATCGC
TTTTCTTTTATTTTTTTTTTTTTTTTTTTTCTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTCTTTTTTTTTTTTTTTTTNTTTNTTTTNTTTNTTTNNNNTNTNTTTTTTGNNNNGTNTANNTNTATNAGAANANNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNGCNNTNNNNTNNTNNNNNNNNNGNGGTTGTTGGTGNNNNNN
+
1111113313333B310A00AAE//A///>01@111>///<//>/////-<-----:-9--9-----;-9----//////-;-9---9-=9-#---#9---#;:-#999####9#-#;----9-####--#-:##:#---#----#-##################################################--##9####9##9#########9#--9--------######
@M01416:220:000000000-DN9DG:1:1101:14672:1775 3:N:0:TTGATCGC
TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTCTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTNTTTNTTTTNTTTNTTTNNNNTNGNGCCCTGGNNNNAANGCNNGNTGGNGGTCNGNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNGANNCNNNNTNNGNNNNNNNNNGNGCGTTGTGTGTNNNNNN
+
11>111>1>0>0A/A/A/>/>/>/>//</////</<------:;----/;0;;B->=9-9-9-9-----9---99-----9--@-;9---9-#-9-#-;--#--9#-;9####-#-#----9--####--#99##9#-;;#;-;-#-##################################################:-##-####-##;#########-#--9--9--9--######
@M01416:220:000000000-DN9DG:1:1101:14624:1784 3:N:0:TTGATCGC
TTTTTTTTCTTTTTTTTTTTTCTTTTCTTTTCCTTCTTTTTCTTTTCTTTTTTTTTTTTTGTGGTTTTTTTTTTTCTTGTTCTTTTTTTCTTNTTTNTTGTNTTTNTTTNNNNTNCNGTCTAGTNNNNGCNCTNNCNGAANTGCANGNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNAGNNGNNNNTNNCNNNNNNNNNGNTGTTTTTGGGTNNNNNN
+
111>1111>B3B1B000A///011112111121B21AB11B0D1112D11D1>E///////00/B/0///////-0111=<111110--/0=#.;.#....#...#.:.####.#.#9999;B0####:;#-9##;#999#9;9-#;##################################################--##9####-##-#########9#---9----9-9######
@M01416:220:000000000-DN9DG:1:1101:14078:1785 3:N:0:TTGATCGC
TTTTTTTTCTTTTTTTTTTTCCTTCTTCCTCCTCTTTTTTTCTCTTTCTTTTTTTTTTTGTTTCTTTCTTTTCTTTTTTTTTTCTTTTCTTTNTTTNTTTTNTTTNTTTNNNNGNCNCCAGTTCNNNNGANCGNNGNGTANGGAANGNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNTCNNTNNNNTNNTNNNNNNNNNTNTGTGTCTTTTTNNNNNN
+
111>11110B3@1B000A//01121BA211100011A11>/012B21221111///>///B002B1122111211B11</>/>0=1111>11#.>.#..<.#...#...####.#.#..::.<0####..#.9##;#;;9#;9..#=##################################################--##;####-##-#########-#----9//9///######
@M01416:220:000000000-DN9DG:1:1101:13957:1791 3:N:0:TTGATCGC
TTTTTTTGTGTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTGTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTNTTTNTTTTNTTTNCTTNNNNTNTNGTGTGGGNNNNGGNAGNNTNGCGNGATGNGNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNGGNNTNNNNTNNGNNNNNNNNNGNTTAGCGGGTGTNNNNNN
+
1111>111B1B0B000A0//A/A/>///>/>//<E//////?000<>-<?-;-:-;--?--;-;-9-@-9----9@-;-9----9-;9-9-@#--9#-9--#9-9#-9-####;#-#;--9---####:-#-;##-#-;-#--;:#-##################################################-;##-####-##-#########-#---;-9---9-######
@M01416:220:000000000-DN9DG:1:1101:15225:1791 3:N:0:TTGATCGC
TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTGTTTTTTTTTTTTTTCTTCTTTTTTTTTTTTTTTTGTTTTTTTTTTTNTTTNTTTGNTTTNTTTNNNNTNGNTCTTTCTNNNNTTNAGNNTNGCGNCGTGNTNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNGGNNGNNNNGNNGNNNNNNNNNGNGGTTTTGGCTGNNNNNN
+
1>>11111>0>0//A/A/>/E/>//>/////<//<-----:--;--/9/;9:-9-;-9-9/;////9;/-;@-9-------;-B----9-9-#-9-#--9-#9--#-;9####-#-#-----//####;-#--##-#---#-9;-#-##################################################-;##-####-##9#########9#-:---9--;9-######
@M01416:220:000000000-DN9DG:1:1101:16193:1792 3:N:0:TTGATCGC
TTTTCTCCGACTTTTTTTTTTCTTCCTTTTCGTTTTTTCGTCTTGTTTGGTTTTTTTGTCCTGCTTGTCTTTTTCGTTCCCTTTCTTTTTTTNTTTNTTTTNTTTNTTTNNNNTNTNTTTTTTGNNNNTTNTTNNTNTTCNAATANANNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNGGNNGNNNNGNNTNNNNNNNNNGNGTGTTGTGTTTNNNNNN
+
11>1>131111113B1A000/01121122B2/00////0/B//AAB100///0///>/B21F11111B1B221>0/00001B11121111>/#///#?///#/?/#/<?####<#<#<....--####.<#<.##.#.:.#....#.##################################################9-##9####-##9#########9#:--;---99/;######
@M01416:220:000000000-DN9DG:1:1101:15141:1793 3:N:0:TAGATCGC
TTTTCTTGCTTTTTTTTTTTCTTTTTTTTTGTCTTTTCTTTGTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTGTTTTTTTTTTTTTCTTNTTTNTTCTNTTTNTTTNNNNTNGNTCTCTGTNNNNTANGGNNGNTGTNGCTTNGNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNGTNNCNNNNGNNGNNNNNNNNNTNTTGTCAGTGTGNNNNNN
+
11>>1B311BD31A1A0AAA011111/////B222B12111120B>//>>/////@<CC/@/@@----:----;--;//;.--;-9--;///#9;;#-;--#9-9#---####-#:#-----//####--#--##-#:9-#--9:#;##################################################99##-####-##-#########:#;:;A-//////######
@M01416:220:000000000-DN9DG:1:1101:17575:1798 3:N:0:TTGATCGC
TTTTCTTTACTTTTTTTTTTTTTTTCTTTTTTTTTTTTTTTTTTGTTTGTTTTTTTTTTTCTTTTCTTTTTTTTTTTTTTCTTTCTTTTTTTNTTTNTTTTNTTTNTTTNNNNTNTNTTGTGTGNNNNCCNCTNNTNTAGNGATANANNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNGTNNTNNNNGNNTNNNNNNNNNCNTTGTTTTTCTTNNNNNN
+
11>11B3B31333311000AAAE//01DAA1//>//>//>///>/B?000??0/>/<---/=<000<=0=0------9--/00//9/9/:9;#--9#;---#-:-#-:9####;#-#--9:---####--#--##;#;9-#;9-;#-##################################################9-##-####-##-#########-#--9-----///######
@M01416:220:000000000-DN9DG:1:1101:14363:1798 3:N:0:TTGATCGC
TTTTTTTTTATTTTTTTTTTTTCCTCTTTTCTTTTCCCCTTTTTTTCTTTTTTTTTTTTTTTTTTTTTTTCTTTTTTTTTGTTTTCTTTCTTNTTTNTCCTNTTTNTTTNNNNTNCNGTGGCCCNNNNGANCCNNCNCGGNATCGNANNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNCCNNTNNNNTNNGNNNNNNNNNGNGTAGGGAAAGGNNNNNN
+
1>>1111>>03333300/////011111112D11D211000111//011111////>///>>CC------/0:000---9-//9B0000000#...#.--;#---#---####-#;#-;-;-9-####9-#--##9#;;;#--9A#-##################################################-;##9####9##;#########-#;;-;9E/;---######
@M01416:220:000000000-DN9DG:1:1101:12672:1800 3:N:0:TTGATCGC
TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTNTTTNTTTTNTTTNTTTNNNNTNTNTGTTATTNNNNTTNTANNCNCGCNTGCGNTNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNGANNTNNNNGNNGNNNNNNNNNGNATGTTGGGGGGNNNNNN
+
11>11>1>>0>0/AAAA////>/>>>/<</</</<-----:-:---;A@@------9@-;99@9@?=-9-99--9-999==>@-@9-@=-9-#-99#-9-:#99-#999####-#:#9--;-//####-9#--##-#99-#-;--#-##################################################;-##9####-##-#########-#-;-:-/-----######
@M01416:220:000000000-DN9DG:1:1101:15759:1815 3:N:0:TTGATCGC
TTTCGCGCCGTTTTTTTTTTTTTTTCTGTTCGTTTTTTCTTCTTGTTGGTTTTTTTTGTCCTTCTTTTCTTTTGTGTTTCCTTTCTTTTTTTNTTGNTTTTNTTTNTTTNNNNTNTNTGTTTGGNNNNGTNCCNNTNTTANAAAANANNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNTGNNGNNNNGNNCNNNNNNNNNTNGGCTGTTGTTANNNNNN
+
1>1111111>0000000/AAE////0122D2//0////01B22B1110///0//>/>/B2>F21111B2B11110000022?22121>11>/#...#>...#<<.#.<<####<#.#;..../.####..#.9##;#..9#..;.#.##################################################--##-####-##-#########-#-9-9--//--/######
@M01416:220:000000000-DN9DG:1:1101:14344:1820 3:N:0:TTGATCGC

There are such instances in the remaining reads in R3
@M01416:220:000000000-DN9DG:1:1101:8395:8719 3:N:0:TAGTTCGC
GAATCCATGCATTCCATTCATAAAGGAGCACGCCAGACCGTCGTGTGCGGAAAGAGTGTCCTGCAGGTCTAGCGCGGGCCTTGCTTTACATGNATGNTGGCNCTGNGGCGNNGGNGNAAGTGCGNNNNGTNGGNNTNTAANATCCNGNNNNNNNNNNGNNNNNNNNNNNNNNNNNNCNNNNCNNCCNNANNNNNNNNTTNNTNNNNTNNGNNGTTNGNNTNTTTTTGGTAGTNNNGCN
+

111>11@B113B3333FB33AF111BE000000A0B00EE///BG/1/A//A00BF1DG1FA1B100111B1>>F/EC//1>1>1111222#//?#??//#/?/#/?//##??#?#//?/?1/####.<#..##.#...#...<#.##########;##################.####.##9-##-########--##-####-##9##-9-#-##-#-;--9---///###9-#
@M01416:220:000000000-DN9DG:1:1101:6706:8719 3:N:0:TAGATCGC
AAGACCCGTTGTGTGTGCAGATAAGCAGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGTCCTGCAGGTCTAGAGCGGGCCCTGGCAGAAATGNAGGNTGGCNTTCNTGTGNNGGNTNTCGCGGGNNNNGGNGTNNTNTTTNAATANANNNNNNNNNNGNNNNNNNNNNNNNNNNNNCNNNNANNCANNCNNNNNNNNGGNNGNNNNTNNCNNGTTNTNNCNTGTGGGGGGTGNNNTTN

BBBBBFBDB@AAAFGFGGGGGGHHHHHHHHH2E2EEHCGGEGGEEEFFBF3FGCGFHDFGHHEGGCFHFGFBGFHGFFFGCHHHHHFAFF4B#??/#??F/#///#/??/##?/#/#////?>/####..#.>##<#<..#....#<##########.##################.####:##;;##.########..##.####.##.##...#.##.#.;9...-----###.:#
@M01416:220:000000000-DN9DG:1:1101:19369:8719 3:N:0:TAGATCGC
AGCTAGCCGTATAAGGAGGCCTGAAGAGATCGGAAGAGCGTCGTGGAGGGAAAGAGTGTCCTGCAGGTCTAGAGCGGCCCTGGCAGAAATGCNGGCNGGCGNACTNAGTTNNTTNANTGGTGGGNNNNCTNTTNNTNTAANAAAANANNNNNNNNNNGNNNNNNNNNNNNNNNNNNCNNNNCNNCCNNCNNNNNNNNGANNGNNNNGNNTNNTTTNTNNGNGGCTTGTGGCTNNNAAN
+
3AABBFFFBBAFGBGGGGGGGGGC4AEHHHHHGGGGHHHGGGGGGEEHGG1GHHFGHGHHHHHHHHHHHHHHHGHGGGCEHFFFFGGHHHFH#??F#??/?#///#//?/##//#/#?/?/?//####//#<<##.#...#<<<E#<##########.##################;####.##;9##9########;;##.####.##.##.9.#:##.#...9.-./...###;.#
@M01416:220:000000000-DN9DG:1:1101:4184:8719 3:N:0:TAGATCGC
AATGTTGGGTAGACACAAGCATCAAGGATACGCTGCACACGCCCTATCCTCCCTGCTCCGAGTCCTATGGGGGTCTTCTGGGCCACTGACTGNTTTNTGTGNCGGNCTCTNNAGNCNGAACCCCNNNNCCNTGNNCNTCANAACCNANNNNNNNNNNTNNNNNNNNNNNNNNNNNNTNNNNGNNGTNNTNNNNNNNNTTNNTNNNNTNNGNNGAGNGNNTNGGTGGGAAAGGNNNTCN
+
111>ABD111111A111AE10BA1A1A0A1A0A00A011B//AA/011A210/BB/1BFA///0B222110/>>/012B110///?BFF111#>>?#//>/#//?#?/?/##/?#/#?/?///?####??#/<##/#/<.#<...#.##########.##################.####9##..##.########99##-####;##;##---#9##-#;--;A@-/-;-###--#
@M01416:220:000000000-DN9DG:1:1101:18776:8719 3:N:0:TAGATTGC
AAAGGTTGGGACACGCAAGCATCAAGGATCCCCTTCACACTCCCCATCCTCCCTGCTCCGTTTCCGTGGGCGGCCTTCTGGCCCACTGCCTGNCTTNTCCGNAGGNCCCANNGGNCNGAACGCCNNNNCCNTCNNCNTCANAACCNANNNNNNNNNNTNNNNNNNNNNNNNNNNNNTNNNNTNNGTNNGNNNNNNNNTTNNANNNNTNNGNNAGCNGNNTNGTCGGGAAAGANNNTCN
+
111>1B1>1A1>111A00E00F11ABG0F0A0B0FB112B00F//00B011AA0B/11B/A1BB2/1?>>/////?F1BF110?//B11011#/?/#</</#//?#??F/##/<#?#?///?//####<>#..##.#<..#<<.<#<##########;##################.####.##.;##-########;9##-####;##;##---#-##-#-;--9-----9###--#
@M01416:220:000000000-DN9DG:1:1101:10910:8719 3:N:0:TAGATCGC
ATCTTGGTTTCAATCTCAGTTTCGGAGGTTGTATGAATTTGGTTTCTTTCTTGGGCAGGCCAGCAGTTGGTTCGGGACTTTAGATCGGAAGANCGTNGTGTNGGGNAAGANNGTNANGCAGGTCNNNNGCNGGNNCNGGCNGAAANGNNNNNNNNNNTNNNNNNNNNNNNNNNNNNGNNNNGNNGGNNCNNNNNNNNACNNANNNNGNNTNNTGTNTNNTNGTGTTGTCTTTNNNTAN
+
AAABAFBFAFFFGGGFGFGFGGHEFEEEAFGGFFH5BFGHHHHHHHGHHEGFFEGHFDG?FGEG2GGGHHGHHGGGEGHHH4@FGFHGCAEF#??F#??F?#??F#??F/##??#?#?//FGGG####?<#??##?#??F#?/<F#?##########.##################.####.##..##9########..##.####.##.##.;.#.##9#..99A.B/;99###.9#
@M01416:220:000000000-DN9DG:1:1101:20570:8720 3:N:0:TAGATCGC
AAAGTTGGGGCACACAAGCATCAAGGATACCCCTCACACTCCCCATCCTCCCTGCTCCGATTCCGAGGGGGGTCTTCTGGGCCACTGACTGANTTGNGTGTNGGANCCTGNNGGNTNAACCCCGNNNNCANTCNNCNCAANACCTNTNNNNNNNNNNTNNNNNNNNNNNNNNNNNNGNNNNTNNTTNNCNNNNNNNNTCNNTNNNNCNNANNTGCNTNNTNTCGGGAAAGAGNNNCCN
+
3A3>AD5C3AA@EFGECEGFGGBF4F4FFHHFEFECAFHFGFG22F3GFFHA2B1F5AA0BEFDF10>EC/<ACFHFH11F.CCCGC0<D=<#<<.#:;CC#;..#;;A.##9;#:#..9CEGB####;.#.;##.#;9.#.;99#;##########.##################.####.##.9##;########:.##.####.##.##...#.##9#;.9:.-9A;/9###;.#
@M01416:220:000000000-DN9DG:1:1101:18223:8720 3:N:0:TAGATCGC
CAGCCACGGCTGCTCGCCTGTCCTCCCCCTGGGGGCGACCGTGATTAAACGTTTAGTTCTTTGTTCTCGCTTTTTTATGTGTTCTCGTTGCANCGTNCCTGNGTTNCATCNNTTNTNTCGACCTNNNNTTNTANNCNCTCNTGTGNTNNNNNNNNNNCNNNNNNNNNNNNNNNNNNCNNNNGNNCCNNCNNNNNNNNGANNCNNNNCNNCNNGGGNTNNGNCCAAATTCATANNNCCN
+
1A?1111>1A11A1AEEA00111A100B0///A//////>////BB@11@/BF0/11B2BF112B1>2//FE/11/<122220222/</B?1#///#///?#/?/#?//?##?/#.#.<><.>>####<.#..##<#...#....#;##########.##################;####-##-;##;########-9##-####-##;##9--#-##-#99--9/9//9/###;;#
@M01416:220:000000000-DN9DG:1:1101:26484:8720 3:N:0:TAGATCGC
ATCTTGGTTTCAATCTCAGTTTCGGAGGTTGCATGAATTTGGTTTCTTTCTCGGGCAGGCCAGCAGTTGGTTTGGGACTTTAGATCGGAAGANCGTNGTGTNGGGNAAGANNGTNCNGCAGGTCNNNNGCNGGNNCNGGCNGAAANGNNNNNNNNNNGNNNNNNNNNNNNNNNNNNGNNNNGNNGGNNTNNNNNNNNTCNNGNNNNTNNTNNGTTNTNNGNTTGGTTTTTTTNNNCGN
+
A>AAA5FF>FFFGFGGGBGEA5GEE2AE24F2A5FDGG5DGHFHHDGBGGB5EE2EE10E11112A5DFBFH1EFE1?FEGFHGF4FA1F1B#11?#????#??F#??F/##??#/#???FF0?####?/#??##/#??/#??CC#<##########.##################.####.##..##9########..##.####.##.##.9;#.##:#9:;..9.;.--###99#
@M01416:220:000000000-DN9DG:1:1101:24218:8720 3:N:0:TAGATCGC
CTTGGGCGCTGCATGGGTGAGTCAACGGCCCCTGCCCCTCAAGACAAGCAGAAGGCATGCGGGCAGCAGCAGGTAGGCGCCCCACCCCCCCCNCACNTCCTNCCANGCGCNNTGNCNGGGCGATNNNNCCNGGNNTNTGANGCGCNGNNNNNNNNNNCNNNNNNNNNNNNNNNNNNGNNNNTNNGGNNGNNNNNNNNTTNNTNNNNTNNCNNGGGNCNNTNCCCGAGCTGTCNNNCGN
+
1A1AAA111ADA11FFG0AEGF11FBEE0E?////0ABAE011B0000/A00EGHEF1BB//>//?/B>000/011F1>EEC/<///<B/BC#///#///?#.>.#....##.<#<#.<<.C--####;.#.:##.#.:.#.9..#;##########.##################-####-##-9##-########9:##-####-##-##---#-##-#----;--99//###9-#
@M01416:220:000000000-DN9DG:1:1101:19699:8720 3:N:0:TAGATCGC
GCCTAGAGCTGGGCCTAAGAAACTCCCCCAGCCCTGCTCTGTCTGTGCTGCTGTGTGGCCAGAGGTGATGCAGGCACCAGATCGGAAGAGCGNCGTNTTGGNCAANAGTGNNCTNCNGGTCTAGNNNNGGNCCNNGNAGANATGCNGNNNNNNNNNNGNNNNNNNNNNNNNNNNNNGNNNNANNCANNTNNNNNNNNGCNNANNNNGNNANNGGGNGNNCNCTGTTTGTACANNNAAN
+
AAAAAF1BD11BEGGG1ECF1FCF1FGEC0EFF?GFHBGBGBEFHAFB21B11FGA0E/0BA0B/1/12BFB10E/EA0>0@F/E/>>0BF>#??>#>/>?#///#/?//##//#/#?//?FB2####</#??##/#/?/#??</#?##########.##################.####.##..##.########..##.####.##;##---#-##-#---9:/:;///###;-#
@M01416:220:000000000-DN9DG:1:1101:5066:8721 3:N:0:TAGATCGC
CTGGAAGTTAGAAGGAAACAGACCACAGACCTGGTCCCCAAAAGAAATGGAGGCAATAGGTTTTGAGGGGCATGGGGACGGGGTTCAGCCTCNAGGNTCCTNCACNCAACNNAGNCNGTGGCCCNNNNGANCCNNCNCGGNATCGNANNNNNNNNNNGNNNNNNNNNNNNNNNNNNCNNNNCNNCTNNCNNNNNNNNACNNTNNNNTNNGNNCATNGNNTNGTAGGGAAAGGNNNTCN
+

1>1>111B33131B1GFGC1EFEHHC0CGFH0A1A100F0B0A/0BAD11BFG/01FBHH1GAG/FFFCGCCEH/E//E@EE@EFB2FFGE#??F#//?/#??/#??//##/?#?#<?<FF0<####/<#??##?#/>>#<>.>#<##########.##################9####.##;.##.########-;##;####;##;##---#-##-#;;9AAE/--;-###--#

[jcardwel]

I'm seeing the same error, though I'm skipping the PE merge step (no fastq-insertPE input):
Pipeline Name : MPRAflow
Pipeline Version: 2.3.5
Fastq insert : /Schwartz/Schwartz/proj/MPRA/20240319_LH00407_0025_B22HHKLLT3/Plasmid_Ctrl_S1_L002_R1_001.fastq.gz
fastq paired : null
Fastq barcode : /Schwartz/Schwartz/proj/MPRA/20240319_LH00407_0025_B22HHKLLT3/cre_only.fastq.gz
design fasta : /Schwartz/Schwartz/proj/MPRA/20240319_LH00407_0025_B22HHKLLT3/mpra_oligos.fasta
minimum BC cov : 3
map quality : 30
base quality : 30
cigar string : n
min % mapped : 0.5
Output dir : outs
Run name : assoc
Working dir : /home/cardwellj/MPRAflow/work
Container Engine: null
Current home : /home/cardwellj
Current user : cardwellj
Current path : /home/cardwellj/MPRAflow
base directory : /home/cardwellj/MPRAflow
Script dir : /home/cardwellj/MPRAflow
Config Profile : standard

executor > local (15)
[95/ae488f] process > count_bc_nolab [100%] 1 of 1 ✔
[f8/14031f] process > create_BWA_ref [100%] 1 of 1 ✔
[5a/cbe2e6] process > align_BWA_S [100%] 11 of 11 ✔
[3d/7dab8e] process > collect_chunks [100%] 1 of 1 ✔
[6d/5d3399] process > map_element_barcodes [ 0%] 0 of 1
[- ] process > filter_barcodes -
Error executing process > 'map_element_barcodes (assign)'

Caused by:
Process map_element_barcodes (assign) terminated with an error exit status (1)

Command executed:

echo "test assign inputs"
echo 30
echo 30
echo cre_only.fastq.gz
zcat cre_only.fastq.gz | head

echo count_fastq.txt
echo count_merged.txt
cat count_fastq.txt
cat count_merged.txt

python /home/cardwellj/MPRAflow/src/nf_ori_map_barcodes.py /home/cardwellj/MPRAflow cre_only.fastq.gz count_fastq.txt s_merged.bam count_merged.txt assoc 30 30 n

Command exit status:
1

Command output:
test assign inputs
30
30
cre_only.fastq.gz
@barcode1
aggacaattc
+
##########
@Barcode2
aatcaccact
+
##########
@barCode3
agttcatgaa
count_fastq.txt
count_merged.txt
280800
20286799
/home/cardwellj/MPRAflow
cre_only.fastq.gz
s_merged.bam
count_fastq.txt
count_merged.txt
counts
20286799
280800
280800
start
bad pairs: 0 poor quality: 14441481
start

Command error:
paired-end reads: 84%|███████████████████████████████████████████████████████████████████████████████████████████████████████████████████▌ | 17118697/20286799.0 [00:25<00:03, 1026300.14it/s]
paired-end reads: 85%|████████████████████████████████████████████████████████████████████████████████████████████████████████████████████▎ | 17224110/20286799.0 [00:25<00:02, 1034491.69it/s]
paired-end reads: 85%|█████████████████████████████████████████████████████████████████████████████████████████████████████████████████████ | 17327586/20286799.0 [00:25<00:02, 1033269.75it/s]
paired-end reads: 86%|█████████████████████████████████████████████████████████████████████████████████████████████████████████████████████▋ | 17433476/20286799.0 [00:25<00:02, 1040668.39it/s]
paired-end reads: 86%|██████████████████████████████████████████████████████████████���███████████████████████████████████████████████████████▍ | 17537569/20286799.0 [00:25<00:02, 1036193.63it/s]
paired-end reads: 87%|███████████████████████████████████████████████████████████████████████████████████████████████████████████████████████▏ | 17643852/20286799.0 [00:25<00:02, 1043995.91it/s]
paired-end reads: 87%|███████████████████████████████████████████████████████████████████████████████████████████████████████████████████████▊ | 17748282/20286799.0 [00:25<00:02, 1041911.78it/s]
paired-end reads: 88%|████████████████████████████████████████████████████████████████████████████████████████████████████████████████████████▌ | 17852495/20286799.0 [00:25<00:02, 1041797.23it/s]
paired-end reads: 89%|█████████████████████████████████████████████████████████████████████████████████████████████████████████████████████████▎ | 17956690/20286799.0 [00:25<00:02, 1038566.28it/s]
paired-end reads: 89%|█████████████████████████████████████████████████████████████████████████████████████████████████████████████████████████▉ | 18060560/20286799.0 [00:25<00:02, 1034613.93it/s]
paired-end reads: 90%|██████████████████████████████████████████████████████████████████████████████████████████████████████████████████████████▋ | 18166215/20286799.0 [00:26<00:02, 1041096.61it/s]
paired-end reads: 90%|███████████████████████████████████████████████████████████████████████████████████████████████████████████████████████████▍ | 18270344/20286799.0 [00:26<00:01, 1035524.37it/s]
paired-end reads: 91%|████████████████████████████████████████████████████████████████████████████████████████████████████████████████████████████ | 18373917/20286799.0 [00:26<00:01, 1008475.76it/s]
paired-end reads: 91%|█████████████████████████████████████████████████████████████████████████████████████████████████████████████████████████████▋ | 18474934/20286799.0 [00:26<00:01, 984875.22it/s]
paired-end reads: 92%|█████████████████████████████████████████████████████████████████████████████████████████████████████████████████████████████▍ | 18578713/20286799.0 [00:26<00:01, 1000133.87it/s]
paired-end reads: 92%|█████████████████████████████████████████████████████████████████████████████████████████████��████████████████████████████████▏ | 18680741/20286799.0 [00:26<00:01, 1006093.25it/s]
paired-end reads: 93%|██████████████████████████████████████████████████████████████████████████████████████████████████████████████████████████████▊ | 18783770/20286799.0 [00:26<00:01, 1013230.24it/s]
paired-end reads: 93%|███████████████████████████████████████████████████████████████████████████████████████████████████████████████████████████████▌ | 18885222/20286799.0 [00:26<00:01, 1005339.30it/s]
paired-end reads: 94%|████████████████████████████████████████████████████████████████████████████████████████████████████████████████████████████████▏ | 18985860/20286799.0 [00:26<00:01, 1002843.27it/s]
executor > local (15)
[95/ae488f] process > count_bc_nolab [100%] 1 of 1 ✔
[f8/14031f] process > create_BWA_ref [100%] 1 of 1 ✔
[5a/cbe2e6] process > align_BWA_S [100%] 11 of 11 ✔
[3d/7dab8e] process > collect_chunks [100%] 1 of 1 ✔
[6d/5d3399] process > map_element_barcodes [100%] 1 of 1, failed: 1 ✘
[- ] process > filter_barcodes -
Error executing process > 'map_element_barcodes (assign)'

Caused by:
Process map_element_barcodes (assign) terminated with an error exit status (1)

Command executed:

echo "test assign inputs"
echo 30
echo 30
echo cre_only.fastq.gz
zcat cre_only.fastq.gz | head

echo count_fastq.txt
echo count_merged.txt
cat count_fastq.txt
cat count_merged.txt

python /home/cardwellj/MPRAflow/src/nf_ori_map_barcodes.py /home/cardwellj/MPRAflow cre_only.fastq.gz count_fastq.txt s_merged.bam count_merged.txt assoc 30 30 n

Command exit status:
1

Command output:
test assign inputs
30
30
cre_only.fastq.gz
@barcode1
aggacaattc
+
##########
@Barcode2
aatcaccact
+
##########
@barCode3
agttcatgaa
count_fastq.txt
count_merged.txt
280800
20286799
/home/cardwellj/MPRAflow
cre_only.fastq.gz
s_merged.bam
count_fastq.txt
count_merged.txt
counts
20286799
280800
280800
start
bad pairs: 0 poor quality: 14441481
start

Command error:
paired-end reads: 84%|███████████████████████████████████████████████████████████████████████████████████████████████████████████████████▌ | 17118697/20286799.0 [00:25<00:03, 1026300.14it/s]
paired-end reads: 85%|████████████████████████████████████████████████████████████████████████████████████████████████████████████████████▎ | 17224110/20286799.0 [00:25<00:02, 1034491.69it/s]
paired-end reads: 85%|█████████████████████████████████████████████████████████████████████████████████████████████████████████████████████ | 17327586/20286799.0 [00:25<00:02, 1033269.75it/s]
paired-end reads: 86%|█████████████████████████████████████████████████████████████████████████████████████████████████████████████████████▋ | 17433476/20286799.0 [00:25<00:02, 1040668.39it/s]
paired-end reads: 86%|██████████████████████████████████████████████████████████████���███████████████████████████████████████████████████████▍ | 17537569/20286799.0 [00:25<00:02, 1036193.63it/s]
paired-end reads: 87%|███████████████████████████████████████████████████████████████████████████████████████████████████████████████████████▏ | 17643852/20286799.0 [00:25<00:02, 1043995.91it/s]
paired-end reads: 87%|███████████████████████████████████████████████████████████████████████████████████████████████████████████████████████▊ | 17748282/20286799.0 [00:25<00:02, 1041911.78it/s]
paired-end reads: 88%|████████████████████████████████████████████████████████████████████████████████████████████████████████████████████████▌ | 17852495/20286799.0 [00:25<00:02, 1041797.23it/s]
paired-end reads: 89%|█████████████████████████████████████████████████████████████████████████████████████████████████████████████████████████▎ | 17956690/20286799.0 [00:25<00:02, 1038566.28it/s]
paired-end reads: 89%|█████████████████████████████████████████████████████████████████████████████████████████████████████████████████████████▉ | 18060560/20286799.0 [00:25<00:02, 1034613.93it/s]
paired-end reads: 90%|██████████████████████████████████████████████████████████████████████████████████████████████████████████████████████████▋ | 18166215/20286799.0 [00:26<00:02, 1041096.61it/s]
paired-end reads: 90%|███████████████████████████████████████████████████████████████████████████████████████████████████████████████████████████▍ | 18270344/20286799.0 [00:26<00:01, 1035524.37it/s]
paired-end reads: 91%|████████████████████████████████████████████████████████████████████████████████████████████████████████████████████████████ | 18373917/20286799.0 [00:26<00:01, 1008475.76it/s]
paired-end reads: 91%|█████████████████████████████████████████████████████████████████████████████████████████████████████████████████████████████▋ | 18474934/20286799.0 [00:26<00:01, 984875.22it/s]
paired-end reads: 92%|█████████████████████████████████████████████████████████████████████████████████████████████████████████████████████████████▍ | 18578713/20286799.0 [00:26<00:01, 1000133.87it/s]
paired-end reads: 92%|█████████████████████████████████████████████████████████████████████████████████████████████��████████████████████████████████▏ | 18680741/20286799.0 [00:26<00:01, 1006093.25it/s]
paired-end reads: 93%|██████████████████████████████████████████████████████████████████████████████████████████████████████████████████████████████▊ | 18783770/20286799.0 [00:26<00:01, 1013230.24it/s]
paired-end reads: 93%|███████████████████████████████████████████████████████████████████████████████████████████████████████████████████████████████▌ | 18885222/20286799.0 [00:26<00:01, 1005339.30it/s]
paired-end reads: 94%|████████████████████████████████████████████████████████████████████████████████████████████████████████████████████████████████▏ | 18985860/20286799.0 [00:26<00:01, 1002843.27it/s]
paired-end reads: 94%|█████████████████████████████████████████████████████████████████████████████████████████████████████████████████████████████████▊ | 19086219/20286799.0 [00:27<00:01, 988198.17it/s]
paired-end reads: 95%|██████████████████████████████████████████████████████████████████████████████████████████████████████████████████████████████████▌ | 19185140/20286799.0 [00:27<00:01, 976406.82it/s]
paired-end reads: 95%|███████████████████████████████████████████████████████████████████████████████████████████████████████████████████████████████████▏ | 19284941/20286799.0 [00:27<00:01, 982788.51it/s]
paired-end reads: 96%|███████████████████████████████████████████████████████████████████████████████████████████████████████████████████████████████████▉ | 19387405/20286799.0 [00:27<00:00, 994881.30it/s]
paired-end reads: 96%|███████████████████████████████████████████████████████████████████████████████████████████████████████████████████████████████████▋ | 19493042/20286799.0 [00:27<00:00, 1012561.83it/s]
paired-end reads: 97%|████████████████████████████████████████████████████████████████████████████████████████████████████████████████████████████████████▎ | 19596306/20286799.0 [00:27<00:00, 1018381.69it/s]
paired-end reads: 97%|█████████████████████████████████████████████████████████████████████████████████████████████████████████████████████████████████████ | 19701573/20286799.0 [00:27<00:00, 1028429.46it/s]
paired-end reads: 98%|█████████████████████████████████████████████████████████████████████████████████████████████████████████████████████████████████████▋ | 19804515/20286799.0 [00:27<00:00, 1024902.68it/s]
paired-end reads: 98%|██████████████████████████████████████████████████████████████████████████████████████████████████████████████████████████████████████▍ | 19907552/20286799.0 [00:27<00:00, 1026534.87it/s]
paired-end reads: 99%|███████████████████████████████████████████████████████████████████████████████████████████████████████████████████████████████████████▏ | 20012411/20286799.0 [00:27<00:00, 1033052.94it/s]
paired-end reads: 99%|███████████████████████████████████████████████████████████████████████████████████████████████████████████████████████████████████████▊ | 20115762/20286799.0 [00:28<00:00, 1024386.73it/s]
paired-end reads: 100%|████████████████████████████████████████████████████████████████████████████████████████████████████████████████████████████████████████▌| 20218250/20286799.0 [00:28<00:00, 1019430.41it/s]
paired-end reads: 100%|██████████████████████████████████████████████████████████████████████████████████████████████████████████████████████████████████████████| 20286799/20286799.0 [00:28<00:00, 719871.27it/s]

barcodes: 0%| | 0/70200.0 [00:00<?, ?it/s]
barcodes: 64%|████████████████████████████████████████████████████████████████████████████████████████████████▊ | 44703/70200.0 [00:00<00:00, 447025.10it/s]
barcodes: 100%|████████████████████████████████████████████████████████████████████████████████████████████████████████████████████████████████████████████████████████| 70200/70200.0 [00:00<00:00, 453737.34it/s]
Traceback (most recent call last):
File "/home/cardwellj/MPRAflow/src/nf_ori_map_barcodes.py", line 157, in
save_barcodes_per_candidate(coords_to_barcodes, f'{prefix}_barcodes_per_candidate.feather')
File "/home/cardwellj/MPRAflow/src/nf_ori_map_barcodes.py", line 134, in save_barcodes_per_candidate
pd.Series(d, name = 'n_barcodes')
File "/home/cardwellj/MPRAflow/work/conda/mpraflow_py36-1978c54da7aacd41df3c7a4cb7639795/lib/python3.6/site-packages/pandas/core/generic.py", line 5063, in getattr
return object.getattribute(self, name)
File "/home/cardwellj/MPRAflow/work/conda/mpraflow_py36-1978c54da7aacd41df3c7a4cb7639795/lib/python3.6/site-packages/pandas/core/accessor.py", line 171, in get
accessor_obj = self._accessor(obj)
File "/home/cardwellj/MPRAflow/work/conda/mpraflow_py36-1978c54da7aacd41df3c7a4cb7639795/lib/python3.6/site-packages/pandas/core/strings.py", line 1796, in init
self._validate(data)
File "/home/cardwellj/MPRAflow/work/conda/mpraflow_py36-1978c54da7aacd41df3c7a4cb7639795/lib/python3.6/site-packages/pandas/core/strings.py", line 1818, in validate
raise AttributeError("Can only use .str accessor with string "
AttributeError: Can only use .str accessor with string values, which use np.object dtype in pandas

Work dir:
/home/cardwellj/MPRAflow/work/6d/5d3399f7da422d10aa82e749dcd45e

Tip: you can replicate the issue by changing to the process work dir and entering the command bash .command.run

[jcardwel]

Oddly, if I go to the work directory and try to run the python command outside of the nextflow pipeline, it appears to compete without error:
(MPRAflow) [cardwellj@e00 5d3399f7da422d10aa82e749dcd45e]$ python3 /home/cardwellj/MPRAflow_batch_test/MPRAflow/src/nf_ori_map_barcodes.py /home/cardwellj/MPRAflow_batch_test/MPRAflow cre_only.fastq.gz count_fastq.txt s_merged.bam count_merged.txt assoc 30 30 n
/home/cardwellj/MPRAflow_batch_test/MPRAflow
cre_only.fastq.gz
s_merged.bam
count_fastq.txt
count_merged.txt
counts
20286799
280800
filtered barcodes: 0it [00:00, ?it/s]

However, if I manually take that output file to the next, filter_barcodes step, I can't get it to complete:
(MPRAflow) [cardwellj@e00 5d3399f7da422d10aa82e749dcd45e]$ python3 /home/cardwellj/MPRAflow_batch_test/MPRAflow/src/nf_filter_barcodes.py assoc assoc_coords_to_barcodes.pickle assoc_barcodes_per_candidate-no_repeats-no_jackpots.feather 3 0.5 /home/cardwellj/MPRAflow/work/95/ae488f924b13641b61162714bb2d76/label_rmIllegalChars.txt
Traceback (most recent call last):
File "/home/cardwellj/MPRAflow_batch_test/MPRAflow/src/nf_filter_barcodes.py", line 24, in
label_file.columns=['coord','label']
File "/home/cardwellj/.local/lib/python3.8/site-packages/pandas/core/generic.py", line 6002, in setattr
return object.setattr(self, name, value)
File "pandas/_libs/properties.pyx", line 69, in pandas._libs.properties.AxisProperty.set
File "/home/cardwellj/.local/lib/python3.8/site-packages/pandas/core/generic.py", line 730, in _set_axis
self._mgr.set_axis(axis, labels)
File "/home/cardwellj/.local/lib/python3.8/site-packages/pandas/core/internals/managers.py", line 225, in set_axis
self._validate_set_axis(axis, new_labels)
File "/home/cardwellj/.local/lib/python3.8/site-packages/pandas/core/internals/base.py", line 70, in _validate_set_axis
raise ValueError(
ValueError: Length mismatch: Expected axis has 1 elements, new values have 2 elements

[visze]

can you please switch to the MPRAsnakeflow pipeline for processing MPRA data. I do not have the time to support this pipeline too.