gem3-mapper soft clips the methylated first base about gembs

[heathsc]

Dear Bekir, Thank you for the report. Would you have a set of these reads that you could share with us? The mapper should only soft-clip if there are mismatches at the read extremities. If we have some example reads we can hopefully figure out what is happening. Cheers, Simon

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On 24 Jul 2018, at 17:29, berguner ***@***.***> wrote: Hello, In our lab we use Taq1 and Msp1 endonucleases restriction/enrichment for RRBS. These enzymes cut at {T/C}GA and {T/C}GG sites so that most of the reads start with a CpG site. It seems that gem3-mapper soft clips the left-most (reads on forward strand) or right-most (reads on reverse strand) base of these reads if these CpG sites were methylated and not bisulfite converted. As a result, methylation rates at these sites are not called accurately. Please check the IGV screenshot for a comparison between bsmap (top) and Gem3-mapper (bottom) alignments. bscall did not detect any methylation at this site even though the methylation rate is 12/12. <https://user-images.githubusercontent.com/23213155/43148576-e1ca1b68-8f65-11e8-8d43-0da6c1da9df8.png> Best, Bekir — You are receiving this because you are subscribed to this thread. Reply to this email directly, view it on GitHub <#27>, or mute the thread <https://github.com/notifications/unsubscribe-auth/ADHPd-8PkLYg6WuS6NrRnSiujMD5TkKRks5uJz1JgaJpZM4Vc962>.

[berguner]

Dear Simon,

Thank you for your attention. Here is a fastq file with 10k reads.

RRBS_sample_for_gemBS.fq.gz

[heathsc]

Dear Bekir, Can you tell me the version of gemBS that you are using and the analysis parameters? Thanks, Simon

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On 27 Jul 2018, at 11:33, berguner ***@***.***> wrote: Dear Simon, Thank you for your attention. Here is a fastq file 10k reads. RRBS_sample_for_gemBS.fq.gz <https://github.com/heathsc/gemBS/files/2235276/RRBS_sample_for_gemBS.fq.gz> — You are receiving this because you commented. Reply to this email directly, view it on GitHub <#27 (comment)>, or mute the thread <https://github.com/notifications/unsubscribe-auth/ADHPdwBAa4URWAGXbFMdJB9YJvEyITyrks5uKt5_gaJpZM4Vc962>.

[berguner]

Dear Simon,

I was running gemBS v3.0.0. I used the default IHEC configurations for RRBS that was given in the manual. I only changed "left_trim = 0".

Best,
Bekir

[heathsc]

OK thanks - that should have worked. I’ll look into it. Simon

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On 30 Jul 2018, at 10:05, berguner ***@***.***> wrote: Dear Simon, I was running gemBS v3.0.0. I used the default IHEC configurations for RRBS that was given in the manual. I only changed "left_trim = 0". Best, Bekir — You are receiving this because you commented. Reply to this email directly, view it on GitHub <#27 (comment)>, or mute the thread <https://github.com/notifications/unsubscribe-auth/ADHPd24280fbAn_5IGC7nPqZDTngC_Ytks5uLr5ggaJpZM4Vc962>.

[berguner]

Hello Simon,

Did you have the chance to fix this issue?

Best, Bekir

[berguner]

Dear Simon,

We are planning to switch to GemBS for RRBS analysis and this issue is preventing us from moving forward. How soon do you think you can fix this issue?

Best, Bekir

[heathsc]

Dear Bekir, Apologies for the delay and I will get back to this issue now. Simon

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On 13 Sep 2018, at 13:43, berguner ***@***.***> wrote: Dear Simon, We are planning to switch to GemBS for RRBS analysis and this issue is preventing us from moving forward. How soon do you think you can fix this issue? Best, Bekir — You are receiving this because you commented. Reply to this email directly, view it on GitHub <#27 (comment)>, or mute the thread <https://github.com/notifications/unsubscribe-auth/ADHPd9Ap79ueb1GsmwcvNi3ogN-j2tI8ks5uakT-gaJpZM4Vc962>.

[heathsc]

Dear Bekir,

I can't reproduce the problem. When I map the sample file you sent, I see many reads that start with CG and almost all of them are mapping with no clipping. I can't check the region that you displayed in the image above as the file you sent me does not seem to have any reads that map to this region. Could you send me an extract from the BAM file that you used to generate the image, with just reads covering the region of interest?

[berguner]

Hello Simon,

Thanks for looking into this. Here are two BAM files with reads which are affected by the same problem. The sequences were aligned to hg38.

Best,
Bekir

S_439260_B_cells_S41229.zip

[heathsc]

Not sure when this changed, but the version on git does not show this problem. Here is the BAM for these reads, and all of them give full length alignments. Simon

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On 18 Sep 2018, at 15:03, berguner ***@***.***> wrote: Hello Simon, Thanks for looking into this. Here are two BAM files with reads which are affected by the same problem. The sequences were aligned to hg38. Best, Bekir S_439260_B_cells_S41229.zip <https://github.com/heathsc/gemBS/files/2392729/S_439260_B_cells_S41229.zip> — You are receiving this because you commented. Reply to this email directly, view it on GitHub <#27 (comment)>, or mute the thread <https://github.com/notifications/unsubscribe-auth/ADHPd_9yAdLXrfhsCejjmS_vjO0qtfhDks5ucO8EgaJpZM4Vc962>.

[berguner]

Dear Simon,

I cloned and installed the latest version but I still have the same problem. Can you please share the BAM file that you generated? Are there any configuration parameter which would affect this?

Best, Bekir

[heathsc]

In my last reply I sent the BAM I generated for the reads you sent me. No special parameters were used. This is very odd! Simon

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On 9 Oct 2018, at 17:44, berguner ***@***.***> wrote: Dear Simon, I cloned and installed the latest version but I still have the same problem. Can you please share the BAM file that you generated? Are there any configuration parameter which would affect this? Best, Bekir — You are receiving this because you commented. Reply to this email directly, view it on GitHub <#27 (comment)>, or mute the thread <https://github.com/notifications/unsubscribe-auth/ADHPd4y8goiU4jBzV3qHDhZh2Gv3gqRLks5ujMRMgaJpZM4Vc962>.

[berguner]

Sorry but I could not find any bam files in the previous message. Am I missing something?

Bekir

[heathsc]

Maybe attachments are removed when sent through github. I’ll send it to you directly. Simon

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On 9 Oct 2018, at 17:51, berguner ***@***.***> wrote: Sorry but I could not find any bam files in the previous message. Am I missing something? Bekir — You are receiving this because you commented. Reply to this email directly, view it on GitHub <#27 (comment)>, or mute the thread <https://github.com/notifications/unsubscribe-auth/ADHPd2t7VDcMpV5tCOdY5dXK-2G-D_Hmks5ujMXzgaJpZM4Vc962>.

[heathsc]

Could you send me an email address I can use to sned you the BAM file? Simon

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On 9 Oct 2018, at 17:51, berguner ***@***.***> wrote: Sorry but I could not find any bam files in the previous message. Am I missing something? Bekir — You are receiving this because you commented. Reply to this email directly, view it on GitHub <#27 (comment)>, or mute the thread <https://github.com/notifications/unsubscribe-auth/ADHPd2t7VDcMpV5tCOdY5dXK-2G-D_Hmks5ujMXzgaJpZM4Vc962>.

[berguner]

Please send them to berguener[at]cemm.at

Bekir

[berguner]

I figured out that you ran the mapper in non-stranded mode but I was running it in stranded mode. I also changed it to non-stranded mode and the problem disappeared. Is this a normal behavior for stranded libraries?

Bekir