Paired end and Tandem orientation. / Reads map to different ID

[wyim-pgl]

Dear Richard, @richardmleggett

After running Nextclip, I've got report and category divided reads.

I checked number of reads in each category and I found each category reads include Pair end orientation and Tandem orientation reads which defined by "Pairs producing good mappings".

Is it normal?

In Pairs producing bad mappings, can you explain "Reads map to different ID"?

Does it mean R1 and R2 mapping to different contigs?

Thank you.

[richardmleggett]

Hi there,

Yes, it's normal to have paired end and tandem orientation reads in each category. If there is a large percentage, then it's a sign of a bad library.

Reads map to different ID is, as you say, when R1 and R2 map to different contigs.

Thanks,
Richard

[wyim-pgl]

But paired end and tandem orientation reads in each category are in output reads file.

[richardmleggett]

You're right - NextClip runs without a reference, so doesn't know what orientation reads are in. The pipeline runs on the output files from NextClip, using a reference, so can then identify them. I guess the pipeline should offer the option to then remove the PE and tandem reads, but it doesn't yet do that. I'll add it as a feature for a later release.

[wyim-pgl]

Oh alright. Nextclip just run with adaptor trim and analysis and then it maps to reference genome to check the quality.

[MesutOezil]

Hi Richard,

I came across an error below for the first time.

ERROR: nextclip_sam_parse.pl: Can't find /home/xxxxx/yyyyy.nextclip - have you run nextclip_index_reference.pl as per the manual?

I have just installed NextClip in a new system, and am wondering if the latest NextClip version requires a .nextclip file, while previous versions did not need.

Any clue for troubleshooting is welcome. Thank you very much in advance.

Best wishes,

Shigehiro

[richardmleggett]

Hi Shigehiro,

It sounds like it is, as the error message says, that you've not run nextclip_index_reference.pl (section 5.2 of the manual). This generates a .nextclip file for your reference. This is not a new feature.

All the best,
Richard

On 9 Mar 2015, at 06:10, MesutOezil <[email protected]mailto:[email protected]> wrote:

Hi Richard,

I came across an error below for the first time.

ERROR: nextclip_sam_parse.pl: Can't find /home/xxxxx/yyyyy.nextclip - have you run nextclip_index_reference.pl as per the manual?

I have just installed NextClip in a new system, and am wondering if the latest NextClip version requires a .nextclip file, while previous versions did not need.

Any clue for troubleshooting is welcome. Thank you very much in advance.

Best wishes,

Shigehiro

—
Reply to this email directly or view it on GitHubhttps://github.com//issues/15#issuecomment-77805012.

[MesutOezil]

Thank you very much, Richard. Following your suggestion, now I have got it done. In fact, I always run the nextclip pipeline, and still don't understand why I did not have this problem before.

Using this opportunity, I would like to share with you a lab guide including modifications to the illumina's standard protocol for the Next Mate Pair Lib Prep Kit - http://www.clst.riken.jp/phylo/imate.html . We have used NextClip in our protocol, and made attempts to increase the proportion of mates in Category A-C defined by NextClip. I hope it helps.

Shigehiro

On 2015/03/11 22:31, Richard Leggett wrote:

Hi Shigehiro,

It sounds like it is, as the error message says, that you've not run nextclip_index_reference.pl (section 5.2 of the manual). This generates a .nextclip file for your reference. This is not a new feature.

All the best,
Richard

On 9 Mar 2015, at 06:10, MesutOezil <[email protected]mailto:[email protected]> wrote:

Hi Richard,

I came across an error below for the first time.

ERROR: nextclip_sam_parse.pl: Can't find /home/xxxxx/yyyyy.nextclip - have you run nextclip_index_reference.pl as per the manual?

I have just installed NextClip in a new system, and am wondering if the latest NextClip version requires a .nextclip file, while previous versions did not need.

Any clue for troubleshooting is welcome. Thank you very much in advance.

Best wishes,

Shigehiro

—
Reply to this email directly or view it on GitHubhttps://github.com//issues/15#issuecomment-77805012.

—
Reply to this email directly or view it on GitHub #15 (comment).

[richardmleggett]

Hi Shigehiro,

Many thanks for sending the link. I've passed it on to Matt Clark, one of my co-authors, who is more of a lab person than me! Looks interesting!

Thanks,
Richard

On 14 Mar 2015, at 09:49, MesutOezil <[email protected]mailto:[email protected]> wrote:

Thank you very much, Richard. Following your suggestion, now I have got it done. In fact, I always run the nextclip pipeline, and still don't understand why I did not have this problem before.

Using this opportunity, I would like to share with you a lab guide including modifications to the illumina's standard protocol for the Next Mate Pair Lib Prep Kit - http://www.clst.riken.jp/phylo/imate.html . We have used NextClip in our protocol, and made attempts to increase the proportion of mates in Category A-C defined by NextClip. I hope it helps.

Shigehiro

On 2015/03/11 22:31, Richard Leggett wrote:

Hi Shigehiro,

It sounds like it is, as the error message says, that you've not run nextclip_index_reference.pl (section 5.2 of the manual). This generates a .nextclip file for your reference. This is not a new feature.

All the best,
Richard

On 9 Mar 2015, at 06:10, MesutOezil <[email protected]mailto:[email protected]mailto:[email protected]> wrote:

Hi Richard,

I came across an error below for the first time.

ERROR: nextclip_sam_parse.pl: Can't find /home/xxxxx/yyyyy.nextclip - have you run nextclip_index_reference.pl as per the manual?

I have just installed NextClip in a new system, and am wondering if the latest NextClip version requires a .nextclip file, while previous versions did not need.

Any clue for troubleshooting is welcome. Thank you very much in advance.

Best wishes,

Shigehiro

—
Reply to this email directly or view it on GitHubhttps://github.com//issues/15#issuecomment-77805012.

—
Reply to this email directly or view it on GitHub #15 (comment).

—
Reply to this email directly or view it on GitHubhttps://github.com//issues/15#issuecomment-80236861.