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using Mothur to demultiplex plate prepared using RLMIDs NaN_ 454 Pyrosequencing 1 10-01-2012 08:34 PM

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Old 12-06-2012, 01:30 PM   #1
Anthony.287
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Location: Ohio

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Default RLMIDs vs GSMIDs

Hello all!
I'm finally starting to get the hang of sfffile, and am finding some things that I can't explain. For instance, one particular region (a pool of 4 RLMID tagged rapid libraries) on the most recent PTP had 43,949 reads. 43,792 of those got sorted in sff's based on the RLMIDs. Out of curiosity, I sorted using the GSMIDs, and ~15,000 were sorted then. What could be happening?

[[email protected] ~]$ sfffile -s GSMIDs /data/R_2012_11_29_16_12_43_FLX05100626_Administrator_Shotgun/sff/HYPJBHH12_Shotgun.sff
Reading the input SFF file(s)...
Generating the split SFF file(s)...
MID1: 3 reads written into the SFF file.
MID2: 0 reads found.
MID3: 13 reads written into the SFF file.
MID4: 10898 reads written into the SFF file.
MID5: 4660 reads written into the SFF file.
MID6: 0 reads found.
MID7: 0 reads found.
MID8: 0 reads found.
MID9: 1 reads written into the SFF file.
MID10: 0 reads found.
MID11: 0 reads found.
MID12: 0 reads found.
MID13: 0 reads found.
MID14: 0 reads found.
[[email protected] ~]$ sfffile -s RLMIDs /data/R_2012_11_29_16_12_43_FLX05100626_Administrator_Shotgun/sff/HYPJBHH12_Shotgun.sff
Reading the input SFF file(s)...
Generating the split SFF file(s)...
RL1: 24344 reads written into the SFF file.
RL2: 11022 reads written into the SFF file.
RL3: 3692 reads written into the SFF file.
RL4: 4734 reads written into the SFF file.
RL5: 0 reads found.
RL6: 1 reads written into the SFF file.
RL7: 0 reads found.
RL8: 0 reads found.
RL9: 0 reads found.
RL10: 0 reads found.
RL11: 0 reads found.
RL12: 0 reads found.


Can the RLMIDs be misinterpreted as GSMIDs, and vice-versa? Any ideas?

Thank you!
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Old 12-10-2012, 05:44 AM   #2
flxlex
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Join Date: Nov 2008
Posts: 415
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Yes, presumably because by default, up to two mismatches are allowed between the MID and the read. If you try allow only for one error, this should not happen. Either copy the Installation_path/454/config/MIDConfig.parse file to a new location and edit it:
Code:
mid = "MID4", "AGCACTGTAG", 2;
becomes
Code:
mid = "MID4", "AGCACTGTAG", 0;
Then run
Code:
sfffile -s GSMIDs -mcf modified_MIDConfig.parse /data/R_2012_11_29_16_12_43_FLX05100626_Administrator_Shotgun/sff/HYPJBHH12_Shotgun.sff
Or to indicate specific MIDs and allow no errors (adding '/0') run
Code:
sfffile -s GSMIDs:mid4/0,mid5/[email protected]/data/R_2012_11_29_16_12_43_FLX05100626_Administrator_Shotgun/sff/HYPJBHH12_Shotgun.sff
sfffile -s RLMIDs:mid1/0,mid2/0,mid3/0,mid4/[email protected]/data/R_2012_11_29_16_12_43_FLX05100626_Administrator_Shotgun/sff/HYPJBHH12_Shotgun.sff
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