not a real issue but a help request

[josemseoane]

Hello,
Firstable, let me thank you for the great job you did here!

I am running the 16S tutorial and I am stuck on filtering reads by quality and length. When I run

read_filter.pl -q 80 -p 20 -l 270 -thread 4 -c both stitched_reads/.assembledfastq

everything goes smoothly but all the filtered fastaq files produced within my filtered_reads folder are empty. I have tried different parameters for -q -p-I but I always get the same.
My read_filter log looks like if i am filtering everything:
file initial qFiltered lFiltered forwardFiltered reverseFiltered final qFilteredPercent lFilteredPercent forwardFilteredPercent reverseFilteredPercent finalPercent
0162532-8513-A_S134_L001.assembled.fastq 177157 177157 0 0 0 0 100.0 0.0 0.0 0.0 0.0
0162533-8514-A_S135_L001.assembled.fastq 118053 118053 0 0 0 0 100.0 0.0 0.0 0.0 0.0
0162534-8515-A_S136_L001.assembled.fastq 171294 171294 0 0 0 0 100.0 0.0 0.0 0.0 0.0
0162535-8516-A_S137_L001.assembled.fastq 120859 120859 0 0 0 0 100.0 0.0 0.0 0.0 0.0
0162536-8517-A_S138_L001.assembled.fastq 143947 143947 0 0 0 0 100.0 0.0 0.0 0.0 0.0
0162537-8518-A_S139_L001.assembled.fastq 150560 150560 0 0 0 0 100.0 0.0 0.0 0.0 0.0
0162538-8519-A_S140_L001.assembled.fastq 228830 228830 0 0 0 0 100.0 0.0 0.0 0.0 0.0
0162539-8520-A_S141_L001.assembled.fastq 172526 172526 0 0 0 0 100.0 0.0 0.0 0.0 0.0
0162540-8521-A_S142_L001.assembled.fastq 202045 202045 0 0 0 0 100.0 0.0 0.0 0.0 0.0
0162541-8522-A_S143_L001.assembled.fastq 150209 150209 0 0 0 0 100.0 0.0 0.0 0.0 0.0
152646A-RPCM0001-515fB_S47_L001.assembled.fastq 27934 27934 0 0 0 0 100.0 0.0 0.0 0.0 0.0
152647A-RPCM0009-515fB_S48_L001.assembled.fastq 8814 8814 0 0 0 0 100.0 0.0 0.0 0.0 0.0
152648A-RPCM0018-515fB_S49_L001.assembled.fastq 14862 14862 0 0 0 0 100.0 0.0 0.0 0.0 0.0
152649A-RPCM0038-515fB_S50_L001.assembled.fastq 7433 7433 0 0 0 0 100.0 0.0 0.0 0.0 0.0
152650A-RPCM0041-515fB_S51_L001.assembled.fastq 13096 13096 0 0 0 0 100.0 0.0 0.0 0.0 0.0
152651A-RPCM0050-515fB_S52_L001.assembled.fastq 21090 21090 0 0 0 0 100.0 0.0 0.0 0.0 0.0
152653A-RPCM0057-515fB_S54_L001.assembled.fastq 21564 21564 0 0 0 0 100.0 0.0 0.0 0.0 0.0
152654A-RPCM0093-515fB_S55_L001.assembled.fastq 6502 6502 0 0 0 0 100.0 0.0 0.0 0.0 0.0
152655A-RPCM0129-515fB_S56_L001.assembled.fastq 23722 23722 0 0 0 0 100.0 0.0 0.0 0.0 0.0
152658A-RPCM0157-515fB_S59_L001.assembled.fastq 4486 4486 0 0 0 0 100.0 0.0 0.0 0.0 0.0
152659A-RPCM0161-515fB_S60_L001.assembled.fastq 6640 6640 0 0 0 0 100.0 0.0 0.0 0.0 0.0
152660A-RPCM0177-515fB_S61_L001.assembled.fastq 1285 1285 0 0 0 0 100.0 0.0 0.0 0.0 0.0
152661A-RPCM0194-515fB_S62_L001.assembled.fastq 8754 8754 0 0 0 0 100.0 0.0 0.0 0.0 0.0
152662A-RPCM0209-515fB_S63_L001.assembled.fastq 16174 16174 0 0 0 0 100.0 0.0 0.0 0.0 0.0
152663A-RPCM0217-515fB_S64_L001.assembled.fastq 2504 2504 0 0 0 0 100.0 0.0 0.0 0.0 0.0
152664A-RPCM0225-515fB_S65_L001.assembled.fastq 2806 2806 0 0 0 0 100.0 0.0 0.0 0.0 0.0
152665A-RPCM0242-515fB_S66_L001.assembled.fastq 1398 1398 0 0 0 0 100.0 0.0 0.0 0.0 0.0
9152652A-RPCM0053-515fB_S53_L001.assembled.fastq 14960 14960 0 0 0 0 100.0 0.0 0.0 0.0 0.0
9152656A-RPCM0138-515fB_S57_L001.assembled.fastq 33840 33840 0 0 0 0 100.0 0.0 0.0 0.0 0.0
9152657A-RPCM0145-515fB_S58_L001.assembled.fastq 33899 33899 0 0 0 0 100.0 0.0 0.0 0.0 0.0

I attach my FastQC report for all my samples combined:

How would you correctly set up the filtering for a set of samples which looks like mine?

Thank you very much in advance for your help and for making this tools available for those not very skilled on programming.

Best regards,

Jose

[gavinmdouglas]

Hey Jose, It looks like your PE reads did not overlap very well in the middle of your stitched reads, which is why you see that extreme dip in quality. Also, I think maybe you’re confusing the “-q” and “-p” options (you can read about FASTQ format here: https://en.wikipedia.org/wiki/FASTQ_format <https://en.wikipedia.org/wiki/FASTQ_format> and type "read_filter.pl —help” for the script documentation). The max quality score is 41 and so 0% of your reads will have a cut-off of 80. A quality cut-off of ~30 for >80% of sites would be reasonable, but there is no one right answer. Cheers, Gavin

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On Feb 21, 2017, at 12:47 PM, josemseoane ***@***.***> wrote: Hello, Firstable, let me thank you for the great job you did here! I am running the 16S tutorial and I am stuck on filtering reads by quality and length. When I run read_filter.pl -q 80 -p 20 -l 270 -thread 4 -c both stitched_reads/.assembledfastq everything goes smoothly but all the filtered fastaq files produced within my filtered_reads folder are empty. I have tried different parameters for -q -p-I but I always get the same. My read_filter log looks like if i am filtering everything: file initial qFiltered lFiltered forwardFiltered reverseFiltered final qFilteredPercent lFilteredPercent forwardFilteredPercent reverseFilteredPercent finalPercent 0162532-8513-A_S134_L001.assembled.fastq 177157 177157 0 0 0 0 100.0 0.0 0.0 0.0 0.0 0162533-8514-A_S135_L001.assembled.fastq 118053 118053 0 0 0 0 100.0 0.0 0.0 0.0 0.0 0162534-8515-A_S136_L001.assembled.fastq 171294 171294 0 0 0 0 100.0 0.0 0.0 0.0 0.0 0162535-8516-A_S137_L001.assembled.fastq 120859 120859 0 0 0 0 100.0 0.0 0.0 0.0 0.0 0162536-8517-A_S138_L001.assembled.fastq 143947 143947 0 0 0 0 100.0 0.0 0.0 0.0 0.0 0162537-8518-A_S139_L001.assembled.fastq 150560 150560 0 0 0 0 100.0 0.0 0.0 0.0 0.0 0162538-8519-A_S140_L001.assembled.fastq 228830 228830 0 0 0 0 100.0 0.0 0.0 0.0 0.0 0162539-8520-A_S141_L001.assembled.fastq 172526 172526 0 0 0 0 100.0 0.0 0.0 0.0 0.0 0162540-8521-A_S142_L001.assembled.fastq 202045 202045 0 0 0 0 100.0 0.0 0.0 0.0 0.0 0162541-8522-A_S143_L001.assembled.fastq 150209 150209 0 0 0 0 100.0 0.0 0.0 0.0 0.0 152646A-RPCM0001-515fB_S47_L001.assembled.fastq 27934 27934 0 0 0 0 100.0 0.0 0.0 0.0 0.0 152647A-RPCM0009-515fB_S48_L001.assembled.fastq 8814 8814 0 0 0 0 100.0 0.0 0.0 0.0 0.0 152648A-RPCM0018-515fB_S49_L001.assembled.fastq 14862 14862 0 0 0 0 100.0 0.0 0.0 0.0 0.0 152649A-RPCM0038-515fB_S50_L001.assembled.fastq 7433 7433 0 0 0 0 100.0 0.0 0.0 0.0 0.0 152650A-RPCM0041-515fB_S51_L001.assembled.fastq 13096 13096 0 0 0 0 100.0 0.0 0.0 0.0 0.0 152651A-RPCM0050-515fB_S52_L001.assembled.fastq 21090 21090 0 0 0 0 100.0 0.0 0.0 0.0 0.0 152653A-RPCM0057-515fB_S54_L001.assembled.fastq 21564 21564 0 0 0 0 100.0 0.0 0.0 0.0 0.0 152654A-RPCM0093-515fB_S55_L001.assembled.fastq 6502 6502 0 0 0 0 100.0 0.0 0.0 0.0 0.0 152655A-RPCM0129-515fB_S56_L001.assembled.fastq 23722 23722 0 0 0 0 100.0 0.0 0.0 0.0 0.0 152658A-RPCM0157-515fB_S59_L001.assembled.fastq 4486 4486 0 0 0 0 100.0 0.0 0.0 0.0 0.0 152659A-RPCM0161-515fB_S60_L001.assembled.fastq 6640 6640 0 0 0 0 100.0 0.0 0.0 0.0 0.0 152660A-RPCM0177-515fB_S61_L001.assembled.fastq 1285 1285 0 0 0 0 100.0 0.0 0.0 0.0 0.0 152661A-RPCM0194-515fB_S62_L001.assembled.fastq 8754 8754 0 0 0 0 100.0 0.0 0.0 0.0 0.0 152662A-RPCM0209-515fB_S63_L001.assembled.fastq 16174 16174 0 0 0 0 100.0 0.0 0.0 0.0 0.0 152663A-RPCM0217-515fB_S64_L001.assembled.fastq 2504 2504 0 0 0 0 100.0 0.0 0.0 0.0 0.0 152664A-RPCM0225-515fB_S65_L001.assembled.fastq 2806 2806 0 0 0 0 100.0 0.0 0.0 0.0 0.0 152665A-RPCM0242-515fB_S66_L001.assembled.fastq 1398 1398 0 0 0 0 100.0 0.0 0.0 0.0 0.0 9152652A-RPCM0053-515fB_S53_L001.assembled.fastq 14960 14960 0 0 0 0 100.0 0.0 0.0 0.0 0.0 9152656A-RPCM0138-515fB_S57_L001.assembled.fastq 33840 33840 0 0 0 0 100.0 0.0 0.0 0.0 0.0 9152657A-RPCM0145-515fB_S58_L001.assembled.fastq 33899 33899 0 0 0 0 100.0 0.0 0.0 0.0 0.0 I attach my FastQC report for all my samples combined: <https://cloud.githubusercontent.com/assets/22190130/23174707/822c4d02-f85d-11e6-8e3d-b9d171dd30ce.png> How would you correctly set up the filtering for a set of samples which looks like mine? Thank you very much in advance for your help and for making this tools available for those not very skilled on programming. Best regards, Jose — You are receiving this because you are subscribed to this thread. Reply to this email directly, view it on GitHub <#4>, or mute the thread <https://github.com/notifications/unsubscribe-auth/AL6T01sfVXGMKBsTU9e9fAloz2Mxlf3Zks5rexUbgaJpZM4MHkY1>.

[gavinmdouglas]

If you have further problems you can post on our google group: https://groups.google.com/forum/#!forum/microbiome-helper

[josemseoane]

Thank you Gavin, I think I got it! Best, Jose El 21 feb 2017, a las 22:34, Gavin Douglas <notifications@github.com<mailto:notifications@github.com>> escribió: If you have further problems you can post on our google group: https://groups.google.com/forum/#!forum/microbiome-helper — You are receiving this because you authored the thread. Reply to this email directly, view it on GitHub<#4 (comment)>, or mute the thread<https://github.com/notifications/unsubscribe-auth/AVKYMgNHCdtGMVxUzZMVwHxc9NZYE0zuks5re1gZgaJpZM4MHkY1>.

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