scFvMiner Files

These scripts can be used for deep sequencing analysis of the scFv antibodies from a synthetic antibody library and
yields complete sequence information on the randomized areas of antibodies enriched from the library by phage display.
The principles of these scripts were described in
# Lövgen, J., Pursiheimo, J.P., Pyykkö, M., Salmi, J. & Lamminmäki, U. (2016) 
# Next generation sequencing of all variable loops of synthetic single 
# framework scFv – application in anti-HDL antibody selections.
# New Biotechnology 33:790-796.

The scripts were released as part of the methods described in to come publication, and are made available to the community 
for non-commercial purposes. The software is provided "as is," with no implied support. For those interested in
contributing code to the package, the authors can be reached at jsalmi <at> btk <dot> fi and mjtpyy <at> utu <dot> fi.


Dependencies
============

The scripts need Java JRE and for modification the Java JDK is required in order to compile the scripts.

The scFvMiner script requires AminoAcid class which can be found from http://www.hannohinsch.com/javagene.html
Include the AminoAcid class in the scFvMiner/bin folder.

The scripts can be used with trimming software such as Trim Galore! and Illumina aliqning tools such as PANDAseq
but they are not required.


Installation
============
The package can be unpacked to where ever wanted and creates directory structure as follows

scFvMiner/bin/				<-- script directory where the scripts can be run
		/test/				<-- example sequences to test the scripts
		/README.txt			<-- this readme file
		
In order to run the scripts Java jre need to be added to System environment path
(if you need to compile add also the jdk bin path):

Windows XP:
	- Right-click My Computer and click Properties
	- In the System Properties window, click on the Advanced tab
	- In the "Advanced" section, click the Environment Variables button
	- Under System Variables, select Path, then click Edit
	- To the end of the line add path (separated with semicolon) to the Java bin-folder ex. "C:\Program Files\Java\jre1.8.0_91\bin". Click OK
Windows 7:
	- Open System properties from Start -> Control Panel -> System and Security -> System -> Advanced system settings
	- From Advanced tab click Environment Variables button
	- Under System Variables, select Path, then click Edit
	- To the end of the line add path (separated with semicolon) to the Java bin-folder ex. "C:\Program Files (x86)\Java\jre1.8.0_91\bin". Click OK
Windows 8 / 10:
	- From the Desktop, right-click the very bottom left corner of the screen to get the Power User Task Menu
	- From the Power User Task Menu, click System
	- Click the Advanced System Settings link in the left column
	- In the System Properties window, click on the Advanced tab, then click the Environment Variables button near the bottom of that tab
	- Under System Variables, select Path, then click Edit
	- To the end of the line add path (separated with semicolon) to the Java bin-folder ex. "C:\Program Files (x86)\Java\jre1.8.0_91\bin". Click OK
Ubuntu/Linux:
	- Open terminal
	- Type "cd ~" and hit enter
	- Type "gedit .bashrc"
	- To the end of the file insert two lines "export JAVA_HOME=/usr/java/<your version of java>" and "export PATH=${PATH}:${JAVA_HOME}/bin"
	- Save and close the file
	- Type "source .bashrc" and hit enter

	
Description
============
The scripts are all located in the folder scFvMiner/bin.

bin:scripts availabe in the package
	combiner.class		combines the forward and reverse read files (.fastq) from Illumina sequencing
	combiner.java		source for combiner
	scFvMiner.class		mines the sequences
	scFvMiner.java		source for scFvMiner
	Follow_multi.class	combines the results from files mined with scFvMiner
	Follow_multi.java	source for Follow_multi
	
	inputsequence.txt	contains the pre- and postfixes for scFvMiner to use in the mining

To compile the .java file type (with java jdk installed) javac filename.java which creates filename.class
	
	
Usage of the scripts
============
Start the command line in Windows (Start->Search/Run->cmd) or Terminal in Ubuntu/Linux.
Navigate to the scFvMiner/bin folder

combiner (can be used instead of aligning tools such as PANDAseq)
	- Usage: java combiner --forward <input forward .fastq file path> --reverse <input reverse .fastq file path>
	- Creates a file where forward and the reverse reads are combined
	- The file is named with forward file name ending with "_com.txt"

scFvMiner
	- Usage: java scFvMiner --fasta <input .fastq_com.txt file> --inputsequence <input affix sequences file> [--errors]
	- Uses the pre and post affixes from inputsequence.txt file
	- Creates file with "_out.txt" file containing the found sequences and their amounts from the input .fastq file
	- With the flag --errors scFvMiner can also be used to search where affix errors are found
	
Follow_multi
	- Usage: java Follow_multi --dir <path to directory of _out.txt-files> [-n] [--limit <number>]
	- Creates .follow (or .follow_normalized) file which sums up all the found sequences
	- Flag -n can be used to normalize the found sequences by the first file in the folder (creates file with .follow_normalized ending)
	- Flag --limit <number> can be used to put a miminum amount of found sequences from the files to be listed.
		In default the limit is one but for ex. --limit 10 sets so that every sequence listed must be found ten times from the files
	
Example run
============
(Windows)

cd C:\scFvMiner\bin

java combiner --forward C:\scFvMiner\test\test1R1.fastq --reverse C:\scFvMiner\test\test1R2.fastq
java combiner --forward C:\scFvMiner\test\test2R1.fastq --reverse C:\scFvMiner\test\test2R2.fastq
java combiner --forward C:\scFvMiner\test\test3R1.fastq --reverse C:\scFvMiner\test\test3R2.fastq
(these create three combined files: test1R2.fastq_com.txt, test2R2.fastq_com.txt, test3R2.fastq_com.txt)

java scFvMiner --fasta C:\scFvMiner\test\test1R2.fastq_com.txt --inputsequence C:\scFvMiner\test\inputsequence.txt
java scFvMiner --fasta C:\scFvMiner\test\test2R2.fastq_com.txt --inputsequence C:\scFvMiner\test\inputsequence.txt
java scFvMiner --fasta C:\scFvMiner\test\test3R2.fastq_com.txt --inputsequence C:\scFvMiner\test\inputsequence.txt
(create three out files, ending with _out.txt, from combined files which are mined with the affixes from inputsequence.txt)

java scFvMiner --fasta C:\scFvMiner\test\test1R2.fastq_com.txt --inputsequence C:\scFvMiner\test\inputsequence.txt --errors
java scFvMiner --fasta C:\scFvMiner\test\test2R2.fastq_com.txt --inputsequence C:\scFvMiner\test\inputsequence.txt --errors
java scFvMiner --fasta C:\scFvMiner\test\test3R2.fastq_com.txt --inputsequence C:\scFvMiner\test\inputsequence.txt --errors
(create files containing the found errors when mined with the affixes from inputsequence.txt)

java Follow_multi --dir C:\scFvMiner\test\ --limit 10
java Follow_multi --dir C:\scFvMiner\test\ --limit 10 -n
(create files where the found sequences and their amounts are combined together, with -n flag the results are normalized based
on the first file red, in this example with the file test1R2.fastq_com.txt_out.txt)