This tutorial will help you get started with LSC by demonstrating how to error correct 500k sample PacBio long reads with 1 million short reads of length 75bp. If you experience any problems following these steps, please don't hesitate to contact us.
Step 1 - Download and extract the example files
Download the example:
Extract the example to an empty folder of your choice. After extracting the folder should contain the following files and folders:
dn800c9107:example moo$ ls data bin run.cfg
The tutorial will be given with the OSX version. However, the steps are the same for all versions.
Step 2 - Examine the example directory contentsBefore we continue, it will be helpful to learn the purpose of each file in this example. When you run LSC on your data, all of these files can be in separate locations if you wish.
- This is the most important file. It is a text file that contains the path to your sequencer reads and the configuration settings. Please see .cfg file format for details. It is simple to edit and you will need to edit it once for each data-set.
- data directory
- This directory contains all of the sequencer reads in the example. In your case, this directory could be anywhere and it may be read-only. In this example, you have a long read file: LR.fa and a short reads file: SR.fa
- bin directory
- This is directory stores all of the LSC binaries. It is important that all the binaries are in the same location. No installation is required! Simply copy this directory to a location convenient for you.
- temp directory
- This is a temporary directory created during the execution of LSC. The results of
the initial short reads mapping is stored here, so this directory can be quite large.
Note: You can use '-clean_up' option in run.cfg file to limit useful intermediate files that LSC keeps after run-time for later reference.
- output directory
- This is directory stores all the useful output files after executing LSC. It is also created during the execution of LSC
Step 4 - Run LSC on the example data
Only one command is need to to initiate LSC.
Make sure your terminal is pointed to the example folder and type the following in one line:
You should then see some output:
=== Welcome to LSC 0.3 === ['python_path ', ' /usr/bin/python'] ['mode ', ' 0'] ['LR_pathfilename ', ' data/LR.fa'] ['LR_filetype ', ' fa'] ['SR_pathfilename ', ' data/SR.fa'] ['SR_filetype ', ' fa'] ['I_nonredundant ', ' N'] ['Nthread1 ', ' 7'] ['Nthread2 ', ' 7'] ['temp_foldername ', ' temp'] ['output_foldername ', ' output'] ['Lpseudochr ', ' 50000000'] ['LgapInpseudochr ', ' 100'] ['I_RemoveBothTails ', ' Y'] ['MinNumberofNonN ', ' 39'] ['MaxN ', ' 1'] ['clean_up ', ' 0'] ['aligner ', ' bowtie2'] ['novoalign_options ', ' -r All -F FA -n 300 -o sam -o'] ['bwa_options ', ' -n 0.08 -o 10 -e 3 -d 0 -i 0 -M 1 -O 0 -E 1 -N'] ['bowtie2_options ', ' --end-to-end -a -f -L 15 --mp 1,1 --np 1 --rdg 0,1 --rfg 0,1 --score-min L,0,-0.08 --no-unal'] ['razers3_options ', ' -i 92 -mr 0 -of sam'] === sort and uniq SR data === 0:00:08.609253 ===split SR:=== 0:00:08.976453 ===compress SR.aa:=== 0:00:04.194401 finsish genome 0:00:04.264694 0:00:04.273020 0:00:04.327496 finsish genome finsish genome 0:00:04.331644 0:00:04.341139 0:00:04.348802 finsish genome finsish genome finsish genome finsish genome 0:00:14.166704 ===RemoveBothTails in LR:=== 0:00:16.357049 ===compress LR:=== /usr/bin/python ../bin_review_3/FASTA2fa.py temp/Notwotails_LR.fa temp/LR.fa rm temp/Notwotails_LR.fa 0:00:16.831772 ===compress LR:=== /usr/bin/python ../bin_review_3/compress.py -MinNonN=0 -MaxN=10000 fa temp/LR.fa temp/LR.fa. 0:00:08.897705 finsish genome rm temp/LR.fa 0:00:26.352285 ===poolchr LR:=== finsish genome 0:00:26.865437 ===bowtie2 index pseudochr:=== Settings: Output files: "temp/pseudochr_LR.fa.cps.*.bt2" Line rate: 6 (line is 64 bytes) Lines per side: 1 (side is 64 bytes) Offset rate: 4 (one in 16) FTable chars: 10 Strings: unpacked . . . . Total time for backward call to driver() for mirror index: 00:00:16 0:00:57.808296 ===bowtie2 SR.aa.cps:=== 134942 reads; of these: 134942 (100.00%) were unpaired; of these: 115074 (85.28%) aligned 0 times 4408 (3.27%) aligned exactly 1 time 15460 (11.46%) aligned >1 times 14.72% overall alignment rate . . . 16.32% overall alignment rate 0:09:28.006754 ===samParser SR.aa.cps.nav:=== 0:10:10.478844 ===convertNAV SR.aa.cps.nav:=== 0:10:18.218553 ===merge_mapping_file SR.aa.cps.nav.map:=== #####write LR_SR_mapping to file:0:00:00.592847 0:10:20.628160 ===cat SR.aa.cps:=== 0:10:20.809205 ===cat SR.aa.idx:=== 0:10:21.019699 ===cat SR.??.cps.sam :=== 0:10:21.938583 ===cat SR.??.cps.nav :=== 0:10:22.068057 ===split LR_SR.map:=== 0:10:22.390992 ===write LR_SR.map.??_tmp:=== finish loading files 0:00:02.571950 0:00:00.880853 0:00:00.929013 0:00:00.881578 0:00:01.012865 0:00:00.755201 0:00:00.788939 0:00:00.909538 0:10:31.767625 ===correct.py LR_SR.map.??_tmp :=== 0:10:31.794182 0:19:52.385551 ===cat full_LR_SR.map.fa :=== ===cat corrected_LR_SR.map.fa :=== ===cat corrected_LR_SR.map.fq :=== ===cat uncorrected_LR_SR.map.fa :===
At this point, feel free to take a break. After about 3-4 minutes the the mapping and error correction will be completed.
Step 5 - Examining the outputAll of the output from LSC is automatically copied to the "output" directory. After this execution, it should contain:
Each output file is descired on maual page in more details:dnab4167d9:output moo$ ls corrected_LR.fa corrected_LR.fq uncorrected_LR.fa full_LR.fa
- As long as there are short reads (SR) mapped to a long read, this long read can be corrected at the SR-covered regions. (Please see more details from the paper). The sequence from the left-most SR-covered base to the right-most SR-covered base is outputted in the file corrected_LR_SR.map
- Although the terminus sequences are corrected, they are concatenated with their corrected sequence (corrected_LR_SR.map.fa) to be a "full" sequence. Thus, this sequence covers the equivalent length as the raw read and is outputted in the file full_LR_SR.map.fa
- This is the negative control. uncorrected_LR_SR.map.fa contains the left-most SR-covered base to the right-most SR-covered base (equivalent region in corrected_LR_SR.map.fa) but not error corrected. Thus, it is fragments of the raw reads.