Hello, I tried to use NGS: SAM Tools ->flagstat on a BAM files for basic stats, but I got results like you see below. It doesn't seem to be working. Any suggestions? 26584869 in total 0 QC failure 0 duplicates 26584869 mapped (100.00%) 0 paired in sequencing 0 read1 0 read2 0 properly paired (-nan%) 0 with itself and mate mapped 0 singletons (-nan%) 0 with mate mapped to a different chr 0 with mate mapped to a different chr (mapQ>=5) Thanks Slim The information in this e-mail is intended only for the person to whom it is addressed. If you believe this e-mail was sent to you in error and the e-mail contains patient information, please contact the Partners Compliance HelpLine at http://www.partners.org/complianceline . If the e-mail was sent to you in error but does not contain patient information, please contact the sender and properly dispose of the e-mail.
Hi, Slim. My guess is that you used an aligner that outputs only aligned reads (tophat, for example) and that the input was single-ended. If that is the case, then what you see below is exactly as expected. If not, then you might need to be more specific about how you generated the BAM file. Sean On Tue, Apr 5, 2011 at 12:31 PM, Slim Sassi <ssassi@ccib.mgh.harvard.edu> wrote:
Hello, I tried to use NGS: SAM Tools ->flagstat on a BAM files for basic stats, but I got results like you see below. It doesn't seem to be working. Any suggestions? 26584869 in total
0 QC failure 0 duplicates 26584869 mapped (100.00%) 0 paired in sequencing 0 read1 0 read2 0 properly paired (-nan%) 0 with itself and mate mapped 0 singletons (-nan%) 0 with mate mapped to a different chr 0 with mate mapped to a different chr (mapQ>=5)
Thanks Slim
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Sean, You are correct, I did use tophat. Can you or anyone suggest a program for BAM/SAM stats where the alignment was done with tophat Thanks Slim On Apr 5, 2011, at 12:51 PM, Sean Davis wrote:
Hi, Slim.
My guess is that you used an aligner that outputs only aligned reads (tophat, for example) and that the input was single-ended. If that is the case, then what you see below is exactly as expected. If not, then you might need to be more specific about how you generated the BAM file.
Sean
On Tue, Apr 5, 2011 at 12:31 PM, Slim Sassi <ssassi@ccib.mgh.harvard.edu> wrote:
Hello, I tried to use NGS: SAM Tools ->flagstat on a BAM files for basic stats, but I got results like you see below. It doesn't seem to be working. Any suggestions? 26584869 in total
0 QC failure 0 duplicates 26584869 mapped (100.00%) 0 paired in sequencing 0 read1 0 read2 0 properly paired (-nan%) 0 with itself and mate mapped 0 singletons (-nan%) 0 with mate mapped to a different chr 0 with mate mapped to a different chr (mapQ>=5)
Thanks Slim
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On Apr 5, 2011 1:05 PM, "Slim Sassi" <ssassi@ccib.mgh.harvard.edu> wrote:
Sean, You are correct, I did use tophat. Can you or anyone suggest a program
for BAM/SAM stats where the alignment was done with tophat
Slim, What stats do you want to capture? The output you gave for flagstats is correct for single-end tophat alignments. All reads are aligned, none are paired, none are marked as duplicates. Sean
Thanks Slim On Apr 5, 2011, at 12:51 PM, Sean Davis wrote:
Hi, Slim.
My guess is that you used an aligner that outputs only aligned reads (tophat, for example) and that the input was single-ended. If that is the case, then what you see below is exactly as expected. If not, then you might need to be more specific about how you generated the BAM file.
Sean
On Tue, Apr 5, 2011 at 12:31 PM, Slim Sassi <ssassi@ccib.mgh.harvard.edu> wrote:
Hello, I tried to use NGS: SAM Tools ->flagstat on a BAM files for basic stats, but I got results like you see below. It doesn't seem to be working. Any suggestions? 26584869 in total
0 QC failure 0 duplicates 26584869 mapped (100.00%) 0 paired in sequencing 0 read1 0 read2 0 properly paired (-nan%) 0 with itself and mate mapped 0 singletons (-nan%) 0 with mate mapped to a different chr 0 with mate mapped to a different chr (mapQ>=5)
Thanks Slim
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Sean, I only wanted to start collecting stats with flagstats but knew that I needed something else to get everthing needed. I would like to know: % that didn't pass QC % mapped % reads in exons/introns/intergenic regions and then, knowing that this is more complicated, I wanted to measure bias within transcripts (for example 3' versus 5'). Of course I am assuming that there is a consistent bias. Thanks Slim On Apr 5, 2011, at 1:50 PM, Sean Davis wrote:
On Apr 5, 2011 1:05 PM, "Slim Sassi" <ssassi@ccib.mgh.harvard.edu> wrote:
Sean, You are correct, I did use tophat. Can you or anyone suggest a program for BAM/SAM stats where the alignment was done with tophat
Slim,
What stats do you want to capture? The output you gave for flagstats is correct for single-end tophat alignments. All reads are aligned, none are paired, none are marked as duplicates.
Sean
Thanks Slim On Apr 5, 2011, at 12:51 PM, Sean Davis wrote:
Hi, Slim.
My guess is that you used an aligner that outputs only aligned reads (tophat, for example) and that the input was single-ended. If that is the case, then what you see below is exactly as expected. If not, then you might need to be more specific about how you generated the BAM file.
Sean
On Tue, Apr 5, 2011 at 12:31 PM, Slim Sassi <ssassi@ccib.mgh.harvard.edu> wrote:
Hello, I tried to use NGS: SAM Tools ->flagstat on a BAM files for basic stats, but I got results like you see below. It doesn't seem to be working. Any suggestions? 26584869 in total
0 QC failure 0 duplicates 26584869 mapped (100.00%) 0 paired in sequencing 0 read1 0 read2 0 properly paired (-nan%) 0 with itself and mate mapped 0 singletons (-nan%) 0 with mate mapped to a different chr 0 with mate mapped to a different chr (mapQ>=5)
Thanks Slim
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On Tue, Apr 5, 2011 at 2:01 PM, Slim Sassi <ssassi@ccib.mgh.harvard.edu> wrote:
Sean, I only wanted to start collecting stats with flagstats but knew that I needed something else to get everthing needed. I would like to know: % that didn't pass QC
That information is not in the FASTQ files, so it will not be in the BAM files, generally.
% mapped
That information assumes that the aligner writes the the unaligned reads to the BAM file. Tophat does not do that.
% reads in exons/introns/intergenic regions
That is not something that flatstats provides. However, there are a number of other tools that could be coerced to give you this type of information (including Galaxy, probably).
and then, knowing that this is more complicated, I wanted to measure bias within transcripts (for example 3' versus 5'). Of course I am assuming that there is a consistent bias.
For this task, you may have to write some code unless someone else on the list is aware of a package that does this for RNA-seq data.
Thanks Slim
On Apr 5, 2011, at 1:50 PM, Sean Davis wrote:
On Apr 5, 2011 1:05 PM, "Slim Sassi" <ssassi@ccib.mgh.harvard.edu> wrote:
Sean, You are correct, I did use tophat. Can you or anyone suggest a program for BAM/SAM stats where the alignment was done with tophat
Slim,
What stats do you want to capture? The output you gave for flagstats is correct for single-end tophat alignments. All reads are aligned, none are paired, none are marked as duplicates.
Sean
Thanks Slim On Apr 5, 2011, at 12:51 PM, Sean Davis wrote:
Hi, Slim.
My guess is that you used an aligner that outputs only aligned reads (tophat, for example) and that the input was single-ended. If that is the case, then what you see below is exactly as expected. If not, then you might need to be more specific about how you generated the BAM file.
Sean
On Tue, Apr 5, 2011 at 12:31 PM, Slim Sassi <ssassi@ccib.mgh.harvard.edu> wrote:
Hello, I tried to use NGS: SAM Tools ->flagstat on a BAM files for basic stats, but I got results like you see below. It doesn't seem to be working. Any suggestions? 26584869 in total
0 QC failure 0 duplicates 26584869 mapped (100.00%) 0 paired in sequencing 0 read1 0 read2 0 properly paired (-nan%) 0 with itself and mate mapped 0 singletons (-nan%) 0 with mate mapped to a different chr 0 with mate mapped to a different chr (mapQ>=5)
Thanks Slim
The information in this e-mail is intended only for the person to whom it is addressed. If you believe this e-mail was sent to you in error and the e-mail contains patient information, please contact the Partners Compliance HelpLine at http://www.partners.org/complianceline . If the e-mail was sent to you in error but does not contain patient information, please contact the sender and properly dispose of the e-mail.
Hello everyone I observed an issue when flagstat is incorporated in a workflow in which the BAM file it works on is also used by another program (generate pileup for instance) and is NOT the input dataset (generated by sam to bam within the workflow). I tested it with the local job runner and with TORQUE (with the pbs scheduler and Maui). - With the local job runner, it works just fine - With TORQUE I get the following error message: pbs_submit failed, PBS error 15033: No free connections Surprisingly, other non-linear workflows work fine. I only observed this error with flagstat. Moreover, when flagstat is in a linear workflow, it works fine too. Ad if it is non-linear but the input dataset is the bam file flagstat works on, it works fine too. Please find attached one of the test workflow where I found the error. The input dataset is a sam file. Any clue? Cheers, LA
Louise-Amélie Schmitt wrote:
Hello everyone
I observed an issue when flagstat is incorporated in a workflow in which the BAM file it works on is also used by another program (generate pileup for instance) and is NOT the input dataset (generated by sam to bam within the workflow).
I tested it with the local job runner and with TORQUE (with the pbs scheduler and Maui).
- With the local job runner, it works just fine
- With TORQUE I get the following error message: pbs_submit failed, PBS error 15033: No free connections
Hi, This can most likely be fixed by increasing the value of NCONNECTS in the TORQUE source, in src/include/libpbs.h, and recompiling on your TORQUE server. I haven't seen a problem after increasing the value to 20. --nate
Surprisingly, other non-linear workflows work fine. I only observed this error with flagstat. Moreover, when flagstat is in a linear workflow, it works fine too. Ad if it is non-linear but the input dataset is the bam file flagstat works on, it works fine too.
Please find attached one of the test workflow where I found the error. The input dataset is a sam file.
Any clue?
Cheers, LA
{ "a_galaxy_workflow": "true", "annotation": "to see if it fails if not forked", "format-version": "0.1", "name": "test flagstat", "steps": { "0": { "annotation": "", "id": 0, "input_connections": {}, "inputs": [ { "description": "", "name": "Input Dataset" } ], "name": "Input dataset", "outputs": [], "position": { "left": 200, "top": 200 }, "tool_errors": null, "tool_id": null, "tool_state": "{\"name\": \"Input Dataset\"}", "tool_version": null, "type": "data_input", "user_outputs": [] }, "1": { "annotation": "", "id": 1, "input_connections": { "source|input1": { "id": 0, "output_name": "output" } }, "inputs": [], "name": "SAM-to-BAM", "outputs": [ { "name": "output1", "type": "bam" } ], "position": { "left": 274.5, "top": 307 }, "tool_errors": null, "tool_id": "sam_to_bam", "tool_state": "{\"source\": \"{\\\"index_source\\\": \\\"cached\\\", \\\"input1\\\": null, \\\"__current_case__\\\": 0}\", \"__page__\": 0}", "tool_version": "1.1.1", "type": "tool", "user_outputs": [] }, "2": { "annotation": "", "id": 2, "input_connections": { "input1": { "id": 1, "output_name": "output1" } }, "inputs": [], "name": "flagstat", "outputs": [ { "name": "output1", "type": "txt" } ], "position": { "left": 396.5, "top": 445 }, "tool_errors": null, "tool_id": "samtools_flagstat", "tool_state": "{\"__page__\": 0, \"input1\": \"null\"}", "tool_version": "1.0.0", "type": "tool", "user_outputs": [] }, "3": { "annotation": "", "id": 3, "input_connections": { "refOrHistory|input1": { "id": 1, "output_name": "output1" } }, "inputs": [], "name": "Generate pileup", "outputs": [ { "name": "output1", "type": "tabular" } ], "position": { "left": 519, "top": 340 }, "tool_errors": null, "tool_id": "sam_pileup", "tool_state": "{\"__page__\": 0, \"c\": \"{\\\"consensus\\\": \\\"no\\\", \\\"__current_case__\\\": 0}\", \"indels\": \"\\\"no\\\"\", \"refOrHistory\": \"{\\\"input1\\\": null, \\\"reference\\\": \\\"indexed\\\", \\\"__current_case__\\\": 0}\", \"lastCol\": \"\\\"no\\\"\", \"mapCap\": \"\\\"60\\\"\"}", "tool_version": "1.1.1", "type": "tool", "user_outputs": [] } } }
___________________________________________________________ The Galaxy User list should be used for the discussion of Galaxy analysis and other features on the public server at usegalaxy.org. Please keep all replies on the list by using "reply all" in your mail client. For discussion of local Galaxy instances and the Galaxy source code, please use the Galaxy Development list:
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Le 06/04/2011 13:57, Nate Coraor a écrit :
Louise-Amélie Schmitt wrote:
Hello everyone
I observed an issue when flagstat is incorporated in a workflow in which the BAM file it works on is also used by another program (generate pileup for instance) and is NOT the input dataset (generated by sam to bam within the workflow).
I tested it with the local job runner and with TORQUE (with the pbs scheduler and Maui).
- With the local job runner, it works just fine
- With TORQUE I get the following error message: pbs_submit failed, PBS error 15033: No free connections Hi,
This can most likely be fixed by increasing the value of NCONNECTS in the TORQUE source, in src/include/libpbs.h, and recompiling on your TORQUE server. I haven't seen a problem after increasing the value to 20.
--nate
Hi, Sorry this is old but I tried recompiling torque after setting the NCONNECTS to 20 and the issue's still there. But there's more: It doesn't affect only flagstat but other non-linear workflows. One of the two jobs that are submitted when their "father" stopped running triggers the same error: PBS error 15033: No free connections And the best part is that it works fine sometimes. But when it crashes, it's always the same job that crashes. Does anyone have a clue? Cheers, L-A
Surprisingly, other non-linear workflows work fine. I only observed this error with flagstat. Moreover, when flagstat is in a linear workflow, it works fine too. Ad if it is non-linear but the input dataset is the bam file flagstat works on, it works fine too.
Please find attached one of the test workflow where I found the error. The input dataset is a sam file.
Any clue?
Cheers, LA { "a_galaxy_workflow": "true", "annotation": "to see if it fails if not forked", "format-version": "0.1", "name": "test flagstat", "steps": { "0": { "annotation": "", "id": 0, "input_connections": {}, "inputs": [ { "description": "", "name": "Input Dataset" } ], "name": "Input dataset", "outputs": [], "position": { "left": 200, "top": 200 }, "tool_errors": null, "tool_id": null, "tool_state": "{\"name\": \"Input Dataset\"}", "tool_version": null, "type": "data_input", "user_outputs": [] }, "1": { "annotation": "", "id": 1, "input_connections": { "source|input1": { "id": 0, "output_name": "output" } }, "inputs": [], "name": "SAM-to-BAM", "outputs": [ { "name": "output1", "type": "bam" } ], "position": { "left": 274.5, "top": 307 }, "tool_errors": null, "tool_id": "sam_to_bam", "tool_state": "{\"source\": \"{\\\"index_source\\\": \\\"cached\\\", \\\"input1\\\": null, \\\"__current_case__\\\": 0}\", \"__page__\": 0}", "tool_version": "1.1.1", "type": "tool", "user_outputs": [] }, "2": { "annotation": "", "id": 2, "input_connections": { "input1": { "id": 1, "output_name": "output1" } }, "inputs": [], "name": "flagstat", "outputs": [ { "name": "output1", "type": "txt" } ], "position": { "left": 396.5, "top": 445 }, "tool_errors": null, "tool_id": "samtools_flagstat", "tool_state": "{\"__page__\": 0, \"input1\": \"null\"}", "tool_version": "1.0.0", "type": "tool", "user_outputs": [] }, "3": { "annotation": "", "id": 3, "input_connections": { "refOrHistory|input1": { "id": 1, "output_name": "output1" } }, "inputs": [], "name": "Generate pileup", "outputs": [ { "name": "output1", "type": "tabular" } ], "position": { "left": 519, "top": 340 }, "tool_errors": null, "tool_id": "sam_pileup", "tool_state": "{\"__page__\": 0, \"c\": \"{\\\"consensus\\\": \\\"no\\\", \\\"__current_case__\\\": 0}\", \"indels\": \"\\\"no\\\"\", \"refOrHistory\": \"{\\\"input1\\\": null, \\\"reference\\\": \\\"indexed\\\", \\\"__current_case__\\\": 0}\", \"lastCol\": \"\\\"no\\\"\", \"mapCap\": \"\\\"60\\\"\"}", "tool_version": "1.1.1", "type": "tool", "user_outputs": [] } } } ___________________________________________________________ The Galaxy User list should be used for the discussion of Galaxy analysis and other features on the public server at usegalaxy.org. Please keep all replies on the list by using "reply all" in your mail client. For discussion of local Galaxy instances and the Galaxy source code, please use the Galaxy Development list:
http://lists.bx.psu.edu/listinfo/galaxy-dev
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Louise-Amélie Schmitt wrote:
Le 06/04/2011 13:57, Nate Coraor a écrit :
Louise-Amélie Schmitt wrote:
Hello everyone
I observed an issue when flagstat is incorporated in a workflow in which the BAM file it works on is also used by another program (generate pileup for instance) and is NOT the input dataset (generated by sam to bam within the workflow).
I tested it with the local job runner and with TORQUE (with the pbs scheduler and Maui).
- With the local job runner, it works just fine
- With TORQUE I get the following error message: pbs_submit failed, PBS error 15033: No free connections Hi,
This can most likely be fixed by increasing the value of NCONNECTS in the TORQUE source, in src/include/libpbs.h, and recompiling on your TORQUE server. I haven't seen a problem after increasing the value to 20.
--nate
Hi,
Sorry this is old but I tried recompiling torque after setting the NCONNECTS to 20 and the issue's still there.
But there's more: It doesn't affect only flagstat but other non-linear workflows. One of the two jobs that are submitted when their "father" stopped running triggers the same error: PBS error 15033: No free connections
Did you recompile both the client and the server? I am not sure which needs it, possibly both.
And the best part is that it works fine sometimes. But when it crashes, it's always the same job that crashes.
Does anyone have a clue?
Cheers, L-A
Surprisingly, other non-linear workflows work fine. I only observed this error with flagstat. Moreover, when flagstat is in a linear workflow, it works fine too. Ad if it is non-linear but the input dataset is the bam file flagstat works on, it works fine too.
Please find attached one of the test workflow where I found the error. The input dataset is a sam file.
Any clue?
Cheers, LA { "a_galaxy_workflow": "true", "annotation": "to see if it fails if not forked", "format-version": "0.1", "name": "test flagstat", "steps": { "0": { "annotation": "", "id": 0, "input_connections": {}, "inputs": [ { "description": "", "name": "Input Dataset" } ], "name": "Input dataset", "outputs": [], "position": { "left": 200, "top": 200 }, "tool_errors": null, "tool_id": null, "tool_state": "{\"name\": \"Input Dataset\"}", "tool_version": null, "type": "data_input", "user_outputs": [] }, "1": { "annotation": "", "id": 1, "input_connections": { "source|input1": { "id": 0, "output_name": "output" } }, "inputs": [], "name": "SAM-to-BAM", "outputs": [ { "name": "output1", "type": "bam" } ], "position": { "left": 274.5, "top": 307 }, "tool_errors": null, "tool_id": "sam_to_bam", "tool_state": "{\"source\": \"{\\\"index_source\\\": \\\"cached\\\", \\\"input1\\\": null, \\\"__current_case__\\\": 0}\", \"__page__\": 0}", "tool_version": "1.1.1", "type": "tool", "user_outputs": [] }, "2": { "annotation": "", "id": 2, "input_connections": { "input1": { "id": 1, "output_name": "output1" } }, "inputs": [], "name": "flagstat", "outputs": [ { "name": "output1", "type": "txt" } ], "position": { "left": 396.5, "top": 445 }, "tool_errors": null, "tool_id": "samtools_flagstat", "tool_state": "{\"__page__\": 0, \"input1\": \"null\"}", "tool_version": "1.0.0", "type": "tool", "user_outputs": [] }, "3": { "annotation": "", "id": 3, "input_connections": { "refOrHistory|input1": { "id": 1, "output_name": "output1" } }, "inputs": [], "name": "Generate pileup", "outputs": [ { "name": "output1", "type": "tabular" } ], "position": { "left": 519, "top": 340 }, "tool_errors": null, "tool_id": "sam_pileup", "tool_state": "{\"__page__\": 0, \"c\": \"{\\\"consensus\\\": \\\"no\\\", \\\"__current_case__\\\": 0}\", \"indels\": \"\\\"no\\\"\", \"refOrHistory\": \"{\\\"input1\\\": null, \\\"reference\\\": \\\"indexed\\\", \\\"__current_case__\\\": 0}\", \"lastCol\": \"\\\"no\\\"\", \"mapCap\": \"\\\"60\\\"\"}", "tool_version": "1.1.1", "type": "tool", "user_outputs": [] } } } ___________________________________________________________ The Galaxy User list should be used for the discussion of Galaxy analysis and other features on the public server at usegalaxy.org. Please keep all replies on the list by using "reply all" in your mail client. For discussion of local Galaxy instances and the Galaxy source code, please use the Galaxy Development list:
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To manage your subscriptions to this and other Galaxy lists, please use the interface at:
Hi Slim, The tool is working correctly as we have it implemented right now, but there is some room for improvement. We are adding some tuning to our near-term to do list. Thanks for the suggestion! Best, Jen Galaxy team On 4/5/11 9:31 AM, Slim Sassi wrote:
Hello, I tried to use NGS: SAM Tools ->flagstat on a BAM files for basic stats, but I got results like you see below. It doesn't seem to be working. Any suggestions?
26584869 in total
0 QC failure 0 duplicates 26584869 mapped (100.00%) 0 paired in sequencing 0 read1 0 read2 0 properly paired (-nan%) 0 with itself and mate mapped 0 singletons (-nan%) 0 with mate mapped to a different chr 0 with mate mapped to a different chr (mapQ>=5)
Thanks Slim
The information in this e-mail is intended only for the person to whom it is addressed. If you believe this e-mail was sent to you in error and the e-mail contains patient information, please contact the Partners Compliance HelpLine at http://www.partners.org/complianceline . If the e-mail was sent to you in error but does not contain patient information, please contact the sender and properly dispose of the e-mail.
___________________________________________________________ The Galaxy User list should be used for the discussion of Galaxy analysis and other features on the public server at usegalaxy.org. Please keep all replies on the list by using "reply all" in your mail client. For discussion of local Galaxy instances and the Galaxy source code, please use the Galaxy Development list:
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To manage your subscriptions to this and other Galaxy lists, please use the interface at:
-- Jennifer Jackson http://usegalaxy.org http://galaxyproject.org
Hi there, I'm running a local installation on a rocks-cluster (centOs5) on a mounted NFS (Ext4 formatted). While importing a fastq file of roughly 20GB via FTP I received the following error: "[Errno28] No Space left on Device" Any help is appreciated. Thanks, Jan
participants (6)
-
Jan Haas
-
Jennifer Jackson
-
Louise-Amélie Schmitt
-
Nate Coraor
-
Sean Davis
-
Slim Sassi