Linux command line problem

[gwaldbieser]

Thanks for this program! I would prefer to use the Linux command line so that I don't have to transfer fastq files to a Windows machine. I am trying to run the test data on Linux but cannot get the command line to accept the inputs, even when using full path information. I have unzipped the test fastq files and renamed the folder to JGPedigree_Fastq (to remove spaces).

(From within /mnt/data8/MegasatTestData/)
$perl /home/software/MEGASAT/MEGASAT_Genotype.pl /mnt/data8/MegasatTestData/primer-input.txt 2 5 /mnt/data8/MegasatTestData/Pedigree/JGPedigree_Fastq/ /mnt/data8/MegasatTestData/output

Missing command line arguments!

#######################################################################

Need the directory of primer file as the first command-line argument

Need the maximum number of mismatches as the second command-line argument

Need the minimum depth threshold as the third command-line argument

Need the directory of data set folder that contains all the input sequence read files (fastq or fasta) as the fourth command-line argument

Need the directory to save the output folder as the fifth command-line argument

I would appreciate if you would you provide more detail on the expected inputs for the command line.
Geoff

[LuyaoZhan]

Hello, I think you are missing the fourth argument that is the number of processors. This argument is just for doing multiprocessing. If you don't want to use multiple processors, you can type 1 as the fourth argument in the command line. If you have any questions, please let me know. Best wishes Catherine

…

On 4 April 2017 at 13:33, gwaldbieser ***@***.***> wrote: Thanks for this program! I would prefer to use the Linux command line so that I don't have to transfer fastq files to a Windows machine. I am trying to run the test data on Linux but cannot get the command line to accept the inputs, even when using full path information. I have unzipped the test fastq files and renamed the folder to JGPedigree_Fastq (to remove spaces). (From within /mnt/data8/MegasatTestData/) $perl /home/software/MEGASAT/MEGASAT_Genotype.pl /mnt/data8/MegasatTestData/primer-input.txt 2 5 /mnt/data8/MegasatTestData/Pedigree/JGPedigree_Fastq/ /mnt/data8/MegasatTestData/output Missing command line arguments! ####################################################################### Need the directory of primer file as the first command-line argument Need the maximum number of mismatches as the second command-line argument Need the minimum depth threshold as the third command-line argument Need the directory of data set folder that contains all the input sequence read files (fastq or fasta) as the fourth command-line argument Need the directory to save the output folder as the fifth command-line argument I would appreciate if you would you provide more detail on the expected inputs for the command line. Geoff — You are receiving this because you are subscribed to this thread. Reply to this email directly, view it on GitHub <#1>, or mute the thread <https://github.com/notifications/unsubscribe-auth/ALo0dbUMxdNRU7cIqIVYyLmRWmiUZaFRks5rsnDEgaJpZM4MzHVz> .

[gwaldbieser]

Ah, that makes sense now in light of the parameters entered in the Windows GUI but it wasn't obvious in the error messaging. It's working now.
Thanks!

[gwaldbieser]

Initially, I ran the test data which worked as expected. When I tested some of my data only blank genotypes were output. I tracked it down to an error on page 6 of the User's Manual where the column labels for 5' flank and 3' flank were switched. Once I switched those columns in my primer file then everything worked well.

[LuyaoZhan]

Thank you for telling me this error in the User's Manual. I didn't notice before. I will change it to the correct format. Thank you. Best wishes Catherine

…

On 5 April 2017 at 11:33, gwaldbieser ***@***.***> wrote: Initially, I ran the test data which worked as expected. When I tested some of my data only blank genotypes were output. I tracked it down to an error on page 6 of the User's Manual where the column labels for 5' flank and 3' flank were switched. Once I switched those columns in my primer file then everything worked well. — You are receiving this because you commented. Reply to this email directly, view it on GitHub <#1 (comment)>, or mute the thread <https://github.com/notifications/unsubscribe-auth/ALo0dYmEn4QwrhJJyriNwQ3GXWWT1A86ks5rs6ZIgaJpZM4MzHVz> .

[gwaldbieser]

I have looked at the manuscript online and supplemental material and cannot find details on the scoring rules and how the six scoring ratios are defined. Can you provide that information, please?

[LuyaoZhan]

I think we provided the scoring information in the supplemental material. Anyway, I just found a word file (attached) that explains the scoring rules. If you have any questions, please let me know. Thank you! Best wishes Catherine

…

On 5 April 2017 at 13:05, gwaldbieser ***@***.***> wrote: I have looked at the manuscript online and supplemental material and cannot find details on the scoring rules and how the six scoring ratios are defined. Can you provide that information, please? — You are receiving this because you commented. Reply to this email directly, view it on GitHub <#1 (comment)>, or mute the thread <https://github.com/notifications/unsubscribe-auth/ALo0ddUlde9BoEyUJz-LkQdDoGFRfMkHks5rs7vHgaJpZM4MzHVz> .

[gwaldbieser]

Hi Catherine,
I couldn't find it in the supporting information online (Appendices S1-S4).
The MS Word file did not attach. Can you post it to the code page?
Thanks.
Geoff

[LuyaoZhan]

Hi Geoff, Could you send me your email address? I can attach that word file in the email. Best wishes Catherine

…

On 5 April 2017 at 13:36, gwaldbieser ***@***.***> wrote: Hi Catherine, I couldn't find it in the supporting information online (Appendices S1-S4). The MS Word file did not attach. Can you post it to the code page? Thanks. Geoff — You are receiving this because you commented. Reply to this email directly, view it on GitHub <#1 (comment)>, or mute the thread <https://github.com/notifications/unsubscribe-auth/ALo0dZ1AXb7Wd66edk4iTLylCk6FHzVXks5rs8L6gaJpZM4MzHVz> .

[gwaldbieser]

Geoff.Waldbieser@ars.usda.gov

Thanks!

[gwaldbieser]

Hi Catherine, When using MEGASAT, does the MRA have to be a perfect repeat? Geoff From: CatherineZhan [mailto:notifications@github.com] Sent: Wednesday, April 05, 2017 11:42 AM To: beiko-lab/MEGASAT <MEGASAT@noreply.github.com> Cc: Waldbieser, Geoff <Geoff.Waldbieser@ARS.USDA.GOV>; Author <author@noreply.github.com> Subject: Re: [beiko-lab/MEGASAT] Linux command line problem (#1) Hi Geoff, Could you send me your email address? I can attach that word file in the email. Best wishes Catherine

On 5 April 2017 at 13:36, gwaldbieser ***@***.******@***.***>> wrote: Hi Catherine, I couldn't find it in the supporting information online (Appendices S1-S4). The MS Word file did not attach. Can you post it to the code page? Thanks. Geoff — You are receiving this because you commented. Reply to this email directly, view it on GitHub <#1 (comment)>, or mute the thread <https://github.com/notifications/unsubscribe-auth/ALo0dZ1AXb7Wd66edk4iTLylCk6FHzVXks5rs8L6gaJpZM4MzHVz> .

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[LuyaoZhan]

Hi Geoff, The MRA does not need to be perfect repeat. We allow sequencing errors in that repeating area. Thank you. Best wishes Catherine

…

On 10 May 2017 at 18:33, gwaldbieser ***@***.***> wrote: Hi Catherine, When using MEGASAT, does the MRA have to be a perfect repeat? Geoff From: CatherineZhan ***@***.*** Sent: Wednesday, April 05, 2017 11:42 AM To: beiko-lab/MEGASAT ***@***.***> Cc: Waldbieser, Geoff ***@***.***>; Author < ***@***.***> Subject: Re: [beiko-lab/MEGASAT] Linux command line problem (#1) Hi Geoff, Could you send me your email address? I can attach that word file in the email. Best wishes Catherine On 5 April 2017 at 13:36, gwaldbieser ***@***.***<mailto: ***@***.***>> wrote: > Hi Catherine, > I couldn't find it in the supporting information online (Appendices S1-S4). > The MS Word file did not attach. Can you post it to the code page? > Thanks. > Geoff > > — > You are receiving this because you commented. > Reply to this email directly, view it on GitHub > <#1 (comment)>, > or mute the thread > <https://github.com/notifications/unsubscribe-auth/ ALo0dZ1AXb7Wd66edk4iTLylCk6FHzVXks5rs8L6gaJpZM4MzHVz> > . > — You are receiving this because you authored the thread. Reply to this email directly, view it on GitHub<https://github.com/ beiko-lab/MEGASAT#1#issuecomment-291921713>, or mute the thread< https://github.com/notifications/unsubscribe-auth/ASb-WsFahIa1T2Hjj2jkq- NE8q7WXbVUks5rs8RPgaJpZM4MzHVz>. This electronic message contains information generated by the USDA solely for the intended recipients. Any unauthorized interception of this message or the use or disclosure of the information it contains may violate the law and subject the violator to civil or criminal penalties. If you believe you have received this message in error, please notify the sender and delete the email immediately. — You are receiving this because you commented. Reply to this email directly, view it on GitHub <#1 (comment)>, or mute the thread <https://github.com/notifications/unsubscribe-auth/ALo0dWhr5EYmfhp64MBDEZHsamQGRktVks5r4i06gaJpZM4MzHVz> .

[gwaldbieser]

Thanks. I’m trying to track down why some new loci worked and some did not. It must be somewhere in the sequence I selected for the primer file. Does the FF sequence need to include all sequence between the FP and MRA? Likewise, does the RF sequence need to include everything from the MRA to the RP? From: CatherineZhan [mailto:notifications@github.com] Sent: Thursday, May 11, 2017 6:29 AM To: beiko-lab/MEGASAT <MEGASAT@noreply.github.com> Cc: Waldbieser, Geoff <Geoff.Waldbieser@ARS.USDA.GOV>; Author <author@noreply.github.com> Subject: Re: [beiko-lab/MEGASAT] Linux command line problem (#1) Hi Geoff, The MRA does not need to be perfect repeat. We allow sequencing errors in that repeating area. Thank you. Best wishes Catherine

On 10 May 2017 at 18:33, gwaldbieser ***@***.******@***.***>> wrote: Hi Catherine, When using MEGASAT, does the MRA have to be a perfect repeat? Geoff From: CatherineZhan ***@***.*** Sent: Wednesday, April 05, 2017 11:42 AM To: beiko-lab/MEGASAT ***@***.******@***.***>> Cc: Waldbieser, Geoff ***@***.******@***.***>>; Author < ***@***.******@***.***>> Subject: Re: [beiko-lab/MEGASAT] Linux command line problem (#1) Hi Geoff, Could you send me your email address? I can attach that word file in the email. Best wishes Catherine On 5 April 2017 at 13:36, gwaldbieser ***@***.***<mailto:

<mailto:notifications@github.com%3cmailto:%0b>> notifications@github.com<mailto:notifications@github.com>>> wrote:

> Hi Catherine, > I couldn't find it in the supporting information online (Appendices S1-S4). > The MS Word file did not attach. Can you post it to the code page? > Thanks. > Geoff > > — > You are receiving this because you commented. > Reply to this email directly, view it on GitHub > <#1 (comment)>, > or mute the thread > <https://github.com/notifications/unsubscribe-auth/

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[LuyaoZhan]

Yes, the FF will be the sequence between FP and the MRA. Likewise, the RF will be the sequence between the MRA and RP. Catherine

…

On 11 May 2017 at 10:17, gwaldbieser ***@***.***> wrote: Thanks. I’m trying to track down why some new loci worked and some did not. It must be somewhere in the sequence I selected for the primer file. Does the FF sequence need to include all sequence between the FP and MRA? Likewise, does the RF sequence need to include everything from the MRA to the RP? From: CatherineZhan ***@***.*** Sent: Thursday, May 11, 2017 6:29 AM To: beiko-lab/MEGASAT ***@***.***> Cc: Waldbieser, Geoff ***@***.***>; Author < ***@***.***> Subject: Re: [beiko-lab/MEGASAT] Linux command line problem (#1) Hi Geoff, The MRA does not need to be perfect repeat. We allow sequencing errors in that repeating area. Thank you. Best wishes Catherine On 10 May 2017 at 18:33, gwaldbieser ***@***.***<mailto: ***@***.***>> wrote: > Hi Catherine, > When using MEGASAT, does the MRA have to be a perfect repeat? > > Geoff > > From: CatherineZhan ***@***.*** > Sent: Wednesday, April 05, 2017 11:42 AM > To: beiko-lab/MEGASAT ***@***.***<mailto: ***@***.***>> > Cc: Waldbieser, Geoff ***@***.***<mailto: ***@***.***>>; Author < > ***@***.******@***.***>> > Subject: Re: [beiko-lab/MEGASAT] Linux command line problem (#1) > > Hi Geoff, > > Could you send me your email address? I can attach that word file in the > email. > > Best wishes > Catherine > > On 5 April 2017 at 13:36, gwaldbieser ***@***.***<mailto: ***@***.***%3cmailto:%0b>> ***@***.***< ***@***.***>>> wrote: > > > Hi Catherine, > > I couldn't find it in the supporting information online (Appendices > S1-S4). > > The MS Word file did not attach. Can you post it to the code page? > > Thanks. > > Geoff > > > > — > > You are receiving this because you commented. > > Reply to this email directly, view it on GitHub > > <#1 (comment)>, > > or mute the thread > > <https://github.com/notifications/unsubscribe-auth/ <https://github.com/notifications/unsubscribe-auth/%0b>> ALo0dZ1AXb7Wd66edk4iTLylCk6FHzVXks5rs8L6gaJpZM4MzHVz> > > . > > > > > — > You are receiving this because you authored the thread. > Reply to this email directly, view it on GitHub<https://github.com/ <https://github.com/%0b>> beiko-lab/MEGASAT#1#issuecomment-291921713>, or mute the thread< > https://github.com/notifications/unsubscribe-auth/ASb-WsFahIa1T2Hjj2jkq- > NE8q7WXbVUks5rs8RPgaJpZM4MzHVz>. > > > > > This electronic message contains information generated by the USDA solely > for the intended recipients. Any unauthorized interception of this message > or the use or disclosure of the information it contains may violate the law > and subject the violator to civil or criminal penalties. If you believe you > have received this message in error, please notify the sender and delete > the email immediately. > > — > You are receiving this because you commented. > Reply to this email directly, view it on GitHub > <#1 (comment)>, > or mute the thread > <https://github.com/notifications/unsubscribe-auth/ ALo0dWhr5EYmfhp64MBDEZHsamQGRktVks5r4i06gaJpZM4MzHVz> > . > — You are receiving this because you authored the thread. Reply to this email directly, view it on GitHub<https://github.com/ beiko-lab/MEGASAT#1#issuecomment-300761855>, or mute the thread< https://github.com/notifications/unsubscribe-auth/ASb- WlJWpsfYGacf0uRLfirdlbRmezhWks5r4vEFgaJpZM4MzHVz>. — You are receiving this because you commented. Reply to this email directly, view it on GitHub <#1 (comment)>, or mute the thread <https://github.com/notifications/unsubscribe-auth/ALo0dY4h3q27mqkkUuHrPyFwRJ4EB_vtks5r4wpRgaJpZM4MzHVz> .

[gwaldbieser]

The entire sequence? It seems that if I skip some bases between the FP and FF sequences then the genotypes don’t get called, but if I only use ‘X’ for the FF then the genotypes are called. My question is – does the entire 5’ flanking sequence on the input reads need to be accounted for by the FP and FF sequences? From: CatherineZhan [mailto:notifications@github.com] Sent: Thursday, May 11, 2017 8:56 AM To: beiko-lab/MEGASAT <MEGASAT@noreply.github.com> Cc: Waldbieser, Geoff <Geoff.Waldbieser@ARS.USDA.GOV>; Author <author@noreply.github.com> Subject: Re: [beiko-lab/MEGASAT] Linux command line problem (#1) Yes, the FF will be the sequence between FP and the MRA. Likewise, the RF will be the sequence between the MRA and RP. Catherine

On 11 May 2017 at 10:17, gwaldbieser ***@***.******@***.***>> wrote: Thanks. I’m trying to track down why some new loci worked and some did not. It must be somewhere in the sequence I selected for the primer file. Does the FF sequence need to include all sequence between the FP and MRA? Likewise, does the RF sequence need to include everything from the MRA to the RP? From: CatherineZhan ***@***.*** Sent: Thursday, May 11, 2017 6:29 AM To: beiko-lab/MEGASAT ***@***.******@***.***>> Cc: Waldbieser, Geoff ***@***.******@***.***>>; Author < ***@***.******@***.***>> Subject: Re: [beiko-lab/MEGASAT] Linux command line problem (#1) Hi Geoff, The MRA does not need to be perfect repeat. We allow sequencing errors in that repeating area. Thank you. Best wishes Catherine On 10 May 2017 at 18:33, gwaldbieser ***@***.***<mailto:

<mailto:notifications@github.com%3cmailto:%0b>> notifications@github.com<mailto:notifications@github.com>>> wrote:

> Hi Catherine, > When using MEGASAT, does the MRA have to be a perfect repeat? > > Geoff > > From: CatherineZhan ***@***.*** > Sent: Wednesday, April 05, 2017 11:42 AM > To: beiko-lab/MEGASAT ***@***.***<mailto:

<mailto:MEGASAT@noreply.github.com%3cmailto:%0b>> MEGASAT@noreply.github.com<mailto:MEGASAT@noreply.github.com>>>

> Cc: Waldbieser, Geoff ***@***.***<mailto:

<mailto:Geoff.Waldbieser@ARS.USDA.GOV%3cmailto:%0b>> Geoff.Waldbieser@ARS.USDA.GOV<mailto:Geoff.Waldbieser@ARS.USDA.GOV>>>; Author <

> ***@***.******@***.******@***.******@***.***>>> > Subject: Re: [beiko-lab/MEGASAT] Linux command line problem (#1) > > Hi Geoff, > > Could you send me your email address? I can attach that word file in the > email. > > Best wishes > Catherine > > On 5 April 2017 at 13:36, gwaldbieser ***@***.***<mailto:

<mailto:notifications@github.com%3cmailto:%0b>> <mailto:notifications@github.com%3cmailto:%0b>> notifications@github.com<<mailto:notifications@github.com%3c>

***@***.***>>> wrote: > > > Hi Catherine, > > I couldn't find it in the supporting information online (Appendices > S1-S4). > > The MS Word file did not attach. Can you post it to the code page? > > Thanks. > > Geoff > > > > — > > You are receiving this because you commented. > > Reply to this email directly, view it on GitHub > > <#1 (comment)>, > > or mute the thread > > <https://github.com/notifications/unsubscribe-auth/

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<https://github.com/%0b>> <https://github.com/%0b>> beiko-lab/MEGASAT#1#issuecomment-291921713>,

or mute the thread< > https://github.com/notifications/unsubscribe-auth/ASb-WsFahIa1T2Hjj2jkq- > NE8q7WXbVUks5rs8RPgaJpZM4MzHVz>. > > > > > This electronic message contains information generated by the USDA solely > for the intended recipients. Any unauthorized interception of this message > or the use or disclosure of the information it contains may violate the law > and subject the violator to civil or criminal penalties. If you believe you > have received this message in error, please notify the sender and delete > the email immediately. > > — > You are receiving this because you commented. > Reply to this email directly, view it on GitHub > <#1 (comment)>, > or mute the thread > <https://github.com/notifications/unsubscribe-auth/

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[LuyaoZhan]

The reason why the genotypes are called when using "X" for the FF is because script will assume the locus does not have FF and search for the other components. In some cases, if you skip some bases between FP and FF, the genotypes should be called. I would suggest you to include the entire sequence for FF and RF. Catherine

…

On 11 May 2017 at 10:59, gwaldbieser ***@***.***> wrote: The entire sequence? It seems that if I skip some bases between the FP and FF sequences then the genotypes don’t get called, but if I only use ‘X’ for the FF then the genotypes are called. My question is – does the entire 5’ flanking sequence on the input reads need to be accounted for by the FP and FF sequences? From: CatherineZhan ***@***.*** Sent: Thursday, May 11, 2017 8:56 AM To: beiko-lab/MEGASAT ***@***.***> Cc: Waldbieser, Geoff ***@***.***>; Author < ***@***.***> Subject: Re: [beiko-lab/MEGASAT] Linux command line problem (#1) Yes, the FF will be the sequence between FP and the MRA. Likewise, the RF will be the sequence between the MRA and RP. Catherine On 11 May 2017 at 10:17, gwaldbieser ***@***.***<mailto: ***@***.***>> wrote: > Thanks. I’m trying to track down why some new loci worked and some did > not. It must be somewhere in the sequence I selected for the primer file. > Does the FF sequence need to include all sequence between the FP and MRA? > Likewise, does the RF sequence need to include everything from the MRA to > the RP? > > From: CatherineZhan ***@***.*** > Sent: Thursday, May 11, 2017 6:29 AM > To: beiko-lab/MEGASAT ***@***.***<mailto: ***@***.***>> > Cc: Waldbieser, Geoff ***@***.***<mailto: ***@***.***>>; Author < > ***@***.******@***.***>> > Subject: Re: [beiko-lab/MEGASAT] Linux command line problem (#1) > > Hi Geoff, > > The MRA does not need to be perfect repeat. We allow sequencing errors in > that repeating area. > > Thank you. > > Best wishes > Catherine > > > On 10 May 2017 at 18:33, gwaldbieser ***@***.***<mailto: ***@***.***%3cmailto:%0b>> ***@***.***< ***@***.***>>> wrote: > > > Hi Catherine, > > When using MEGASAT, does the MRA have to be a perfect repeat? > > > > Geoff > > > > From: CatherineZhan ***@***.*** > > Sent: Wednesday, April 05, 2017 11:42 AM > > To: beiko-lab/MEGASAT ***@***.***<mailto: ***@***.***%3cmailto:%0b>> ***@***.******@***.***>>> > > Cc: Waldbieser, Geoff ***@***.***<mailto: ***@***.***%3cmailto:%0b>> ***@***.******@***.***>>>; Author < > > ***@***.******@***.***<mailto: ***@***.******@***.***>>> > > Subject: Re: [beiko-lab/MEGASAT] Linux command line problem (#1) > > > > Hi Geoff, > > > > Could you send me your email address? I can attach that word file in the > > email. > > > > Best wishes > > Catherine > > > > On 5 April 2017 at 13:36, gwaldbieser ***@***.***< mailto: ***@***.***%3cmailto:%0b>> <mailto: ***@***.***%3cmailto:%0b>> ***@***.***<<mailto: ***@***.***%3c> > ***@***.***>>> wrote: > > > > > Hi Catherine, > > > I couldn't find it in the supporting information online (Appendices > > S1-S4). > > > The MS Word file did not attach. Can you post it to the code page? > > > Thanks. > > > Geoff > > > > > > — > > > You are receiving this because you commented. > > > Reply to this email directly, view it on GitHub > > > <#1 (comment)- 291920098>, > > > > or mute the thread > > > <https://github.com/notifications/unsubscribe-auth/ <https://github.com/notifications/unsubscribe-auth/%0b>> < https://github.com/notifications/unsubscribe-auth/%0b>> > ALo0dZ1AXb7Wd66edk4iTLylCk6FHzVXks5rs8L6gaJpZM4MzHVz> > > > . > > > > > > > > > — > > You are receiving this because you authored the thread. > > Reply to this email directly, view it on GitHub<https://github.com/ <https://github.com/%0b>> <https://github.com/%0b>> beiko-lab/MEGASAT#1#issuecomment-291921713>, > or mute the thread< > > https://github.com/notifications/unsubscribe- auth/ASb-WsFahIa1T2Hjj2jkq- > > NE8q7WXbVUks5rs8RPgaJpZM4MzHVz>. > > > > > > > > > > This electronic message contains information generated by the USDA > solely > > for the intended recipients. 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