Empty sam file

[mrb20045]

hi
I used bs3 and every things goes well as follow. But at the end sam file is empty. I found out some other people faced to the same problem. I think that the main code has some problem. Let me know can it be my fault.

python '/home/bs3/bs3-align.py' -1 '/home/Data/R1_1.fq.gz' -2 '/home/Data/R1_2.fq.gz' -g '/home/ARS-UCD1/bt_ref_ARS-UCD1.2.fa' -d '/media/reference_genome' -o aex

 BS-Seeker3 beta

Welcome to SNAP version 1.0beta.23.

Loading index from directory... 59s. 961853050 bases, seed size 20
Aligning.
Total Reads Aligned, MAPQ >= 10 Aligned, MAPQ < 10 Unaligned Too Short/Too Many Ns %Pairs Reads/s Time in Aligner (s)
400,000 325,572 (81.39%) 36,032 (9.01%) 27,723 (6.93%) 10,673 (2.67%) 45.33% 78,724 5
(None, None)

BS-seeker3 Result

Final Alignment Report

================================================

Number of reads in total: 400000
Number of unique-hits reads (before post-filtering): 0
Number of reads mapped after post-filtering 0

Alignment Time: 79.7409200668secs

Final Cytosine Report

================================================

Total Number of Cytosines: 0
Total Number of Cs in CpG context: 0
Total Number of Cs in CHG context: 0
Total Number of Cs in CHH context: 0

Rate of Methylation
mCG 0.000%
mCHG 0.000%
mCHH 0.000%

[khuang28jhu]

1. Did the toy example work?
   If yes, Does this help?

2. Maybe try increasing the -m NO_MISMATCHES or -K ALIGNMENT LENGTH number ?

[mrb20045]

Thanks for attention. Toy example works, So I have to try your suggestion.

[mrb20045]

I tried with -m 30, -m 40, -K 50 and -K 90 (my read length is 100). The results are as follow and all of them are same.

-m 30
BS-Seeker3 beta

Welcome to SNAP version 1.0beta.23.

Loading index from directory... 60s. 961853050 bases, seed size 20
Aligning.
Total Reads Aligned, MAPQ >= 10 Aligned, MAPQ < 10 Unaligned Too Short/Too Many Ns %Pairs Reads/s Time in Aligner (s)
400,000 325,572 (81.39%) 36,032 (9.01%) 27,723 (6.93%) 10,673 (2.67%) 45.33% 87,489 5
(None, None)

BS-seeker3 Result

Final Alignment Report

================================================

Number of reads in total: 400000
Number of unique-hits reads (before post-filtering): 0
Number of reads mapped after post-filtering 0

Alignment Time: 79.7702047825secs

Final Cytosine Report

================================================

Total Number of Cytosines: 0
Total Number of Cs in CpG context: 0
Total Number of Cs in CHG context: 0
Total Number of Cs in CHH context: 0

Rate of Methylation
mCG 0.000%
mCHG 0.000%
mCHH 0.000%

-m 40
BS-Seeker3 beta

Welcome to SNAP version 1.0beta.23.

Loading index from directory... 58s. 961853050 bases, seed size 20
Aligning.
Total Reads Aligned, MAPQ >= 10 Aligned, MAPQ < 10 Unaligned Too Short/Too Many Ns %Pairs Reads/s Time in Aligner (s)
400,000 325,572 (81.39%) 36,032 (9.01%) 27,723 (6.93%) 10,673 (2.67%) 45.33% 86,355 5
(None, None)

BS-seeker3 Result

Final Alignment Report

================================================

Number of reads in total: 400000
Number of unique-hits reads (before post-filtering): 0
Number of reads mapped after post-filtering 0

Alignment Time: 78.3472340107secs

Final Cytosine Report

================================================

Total Number of Cytosines: 0
Total Number of Cs in CpG context: 0
Total Number of Cs in CHG context: 0
Total Number of Cs in CHH context: 0

Rate of Methylation
mCG 0.000%
mCHG 0.000%
mCHH 0.000%

-K 50
BS-Seeker3 beta

Welcome to SNAP version 1.0beta.23.

Loading index from directory... 58s. 961853050 bases, seed size 20
Aligning.
Total Reads Aligned, MAPQ >= 10 Aligned, MAPQ < 10 Unaligned Too Short/Too Many Ns %Pairs Reads/s Time in Aligner (s)
400,000 325,572 (81.39%) 36,032 (9.01%) 27,723 (6.93%) 10,673 (2.67%) 45.33% 88,358 5
(None, None)

BS-seeker3 Result

Final Alignment Report

================================================

Number of reads in total: 400000
Number of unique-hits reads (before post-filtering): 0
Number of reads mapped after post-filtering 0

Alignment Time: 77.1407189369secs

Final Cytosine Report

================================================

Total Number of Cytosines: 0
Total Number of Cs in CpG context: 0
Total Number of Cs in CHG context: 0
Total Number of Cs in CHH context: 0

Rate of Methylation
mCG 0.000%
mCHG 0.000%
mCHH 0.000%

-K 90
BS-Seeker3 beta

Welcome to SNAP version 1.0beta.23.

Loading index from directory... 58s. 961853050 bases, seed size 20
Aligning.
Total Reads Aligned, MAPQ >= 10 Aligned, MAPQ < 10 Unaligned Too Short/Too Many Ns %Pairs Reads/s Time in Aligner (s)
400,000 325,572 (81.39%) 36,032 (9.01%) 27,723 (6.93%) 10,673 (2.67%) 45.33% 87,126 5
(None, None)

BS-seeker3 Result

Final Alignment Report

================================================

Number of reads in total: 400000
Number of unique-hits reads (before post-filtering): 0
Number of reads mapped after post-filtering 0

Alignment Time: 78.0903060436secs

Final Cytosine Report

================================================

Total Number of Cytosines: 0
Total Number of Cs in CpG context: 0
Total Number of Cs in CHG context: 0
Total Number of Cs in CHH context: 0

Rate of Methylation
mCG 0.000%
mCHG 0.000%
mCHH 0.000%

[khuang28jhu]

Can you show the the exact process (exact screen printout and command) when you run the toy example from start to finish?

[mrb20045]

./bs3-align -1 test_data/pair1.fq -2 test_data/pair2.fq -o WGBC -f sam -g reference_genome/genome.fa -d reference_genome/

 BS-Seeker3 beta

Welcome to SNAP version 1.0beta.23.

Loading index from directory... 0s. 60856842 bases, seed size 20
Aligning.
Total Reads Aligned, MAPQ >= 10 Aligned, MAPQ < 10 Unaligned Too Short/Too Many Ns %Pairs Reads/s Time in Aligner (s)
20,000 19,281 (96.41%) 719 (3.60%) 0 (0.00%) 0 (0.00%) 47.78% 132,450 0
(None, None)

BS-seeker3 Result

Final Alignment Report

================================================

Number of reads in total: 20000
Number of unique-hits reads (before post-filtering): 9990.0
Number of reads mapped after post-filtering 9919.0

Alignment Time: 1.49422216415secs

Final Cytosine Report

================================================

Total Number of Cytosines: 348024.0
Total Number of Cs in CpG context: 45187.0
Total Number of Cs in CHG context: 49571.0
Total Number of Cs in CHH context: 253266.0

Rate of Methylation
mCG 99.900%
mCHG 99.905%
mCHH 99.898%

[khuang28jhu]

So the toy example Does work then

does this help?

[mrb20045]

Sorry, but I revised my previous comment. Toy example works but dose not work for my real data.

[lihui215]

@mrb20045 I have the same question with you, have you solved it?

[mrb20045]

Unfortunatly no.

…

On Wed, 30 Jan 2019, 06:16 lihui215 ***@***.*** wrote: [image: Boxbe] <https://www.boxbe.com/overview> This message is eligible for Automatic Cleanup! ***@***.***) Add cleanup rule <https://www.boxbe.com/popup?url=https%3A%2F%2Fwww.boxbe.com%2Fcleanup%3Fkey%3DxvzznjneOqmPa31nFNmnubgcrBdG9LuIHrJohYadgZY%253D%26token%3DUJjS2urm%252Fa6WUpuWqp3vHPlw2wVdu6IZ7UHzawSvfg1vENOqrL%252FIAMiCORNWNkXOX0lbJz%252BM8cZ4YvDL9FTgV%252B2rOfRiMA7LTQqOVQC%252FDloBa3KGp9OJ89toKwnuhBCb630swuh3n2oWsdaxPpCYrw%253D%253D&tc_serial=47511721153&tc_rand=796421542&utm_source=stf&utm_medium=email&utm_campaign=ANNO_CLEANUP_ADD&utm_content=001> | More info <http://blog.boxbe.com/general/boxbe-automatic-cleanup?tc_serial=47511721153&tc_rand=796421542&utm_source=stf&utm_medium=email&utm_campaign=ANNO_CLEANUP_ADD&utm_content=001> @mrb20045 <https://github.com/mrb20045> I have the same question with you, have you solved it? — You are receiving this because you were mentioned. Reply to this email directly, view it on GitHub <#10 (comment)>, or mute the thread <https://github.com/notifications/unsubscribe-auth/AJc6ntzmma3aXkRJoL477USDO9ptQs83ks5vIQFsgaJpZM4VfFMh> .

[lihui215]

@mrb20045 thanks, did you chose BS-seeker2 instead of this one ?

[mrb20045]

I use bismark and BS-seeker2. I belie that the combination of bioinformatics software would be better.

…

On Wed, Jan 30, 2019 at 8:36 AM lihui215 ***@***.***> wrote: [image: Boxbe] <https://www.boxbe.com/overview> This message is eligible for Automatic Cleanup! ***@***.***) Add cleanup rule <https://www.boxbe.com/popup?url=https%3A%2F%2Fwww.boxbe.com%2Fcleanup%3Fkey%3D9R0Ojbx7kbahGaLs5sWOvbYnd9B5AdwWwpZ7b69aATs%253D%26token%3DPJD1KYpQRhO1h8NH9u3GyUr0gyKNzLdsRMGrQcvF1dhzLscvzaiBQdxX1R6ppaWAU1FW2TfO3hZxnaxLzb7UKymLwCZCg%252Be69kAIMEuBlX8U8xdTRxx0xolN0V4YMQYQtRSE1D96H8CQD8Z1VbRatw%253D%253D&tc_serial=47514616408&tc_rand=1636182255&utm_source=stf&utm_medium=email&utm_campaign=ANNO_CLEANUP_ADD&utm_content=001> | More info <http://blog.boxbe.com/general/boxbe-automatic-cleanup?tc_serial=47514616408&tc_rand=1636182255&utm_source=stf&utm_medium=email&utm_campaign=ANNO_CLEANUP_ADD&utm_content=001> @mrb20045 <https://github.com/mrb20045> thanks, did you chose BS-seeker2 instead of this one ? — You are receiving this because you were mentioned. Reply to this email directly, view it on GitHub <#10 (comment)>, or mute the thread <https://github.com/notifications/unsubscribe-auth/AJc6nhkHAargzm0jW-y7_IZbRAcfDDhHks5vISAygaJpZM4VfFMh> .

-- Mohammad Reza Bakhtiarizadeh PhD in Genetics and Animal Breeding Department of animal and poultry science College of Aburaihan- University of Tehran- Iran Phone-Fax:+(98) 021-36040907 P.O : 33916-53775 Home page: http://abu.ut.ac.ir/faculty/?ID=mrbakhtiari Google Scholar: https://scholar.google.com/citations?user=ZtpFeUgAAAAJ&hl=en Github: https://github.com/mrb20045 Researchgate: https://www.researchgate.net/profile/Mohammad_Reza_Bakhtiarizadeh​ ORCID iD: 0000-0001-5336-6987