Table of Contents

• Introduction
• Tools
• Installation
• Usage
• Use u4falign for manipulating alignment results
• Difference between read-SAM and frag-SAM

Introduction

Falign is a sequence alignment toolkit for long noisy chromosome conformation capture (3C) reads, such as Pore-C. Falign is written in C and C++ programming language.

Tools

Three tools are released together in this toolkit.

• falign. The alignment tool.
• falign_ngf. Another alignment tool. It is used for benchmark only.
• u4falign. A utility for manipulating SAM or PAF mapping results.
• The directory supplementary_source_code contains scripts for generating simulated Pore-C reads and analyzing mapping results.

Installation

Download files

$ git clones https://github.com/xiaochuanle/Falign.git

Install from executable binaries

$ cd Falign/release
$ tar xzvf Falign-0.0.1-20221010-Linux-amd64.tar.gz
$ cd Falign/Linux-amd64/bin/
$ export PATH=$PATH:$(pwd)

The last command export PATH=$PATH:$(pwd) is used for adding the path of falign to the system PATH so that you don't have to type the full path of falign (such as /data3/cy/map_test/Falign/Linux-amd64/bin/falign) every time you used falign.

Install from source codes

$ cd Falign/src
$ make -j
$ cd ../Linux-amd64/bin/
$ export PATH=$PATH:$(pwd)

Usage

Decompress the sample data and decompress it.

$ cd Falign
$ tar xzvf sample-data.tar.gz
$ cd sample-data

We provide one sample reference ara_2_4.fa in the ./sample-data/ref directory:

$ ls ref
ara_2_4.fa

and provide three sample reads in the ./sample-data/reads/ directory:

$ ls reads
ara_reads_1.fq ara_reads_2.fq ara_reads_3.fq

Quick Start

By default, falign outputs mapping results in SAM format:

$ falign -num_threads 48 ^GATC ref/ara_2_4.fa \
	reads/ara_reads_1.fq \
    reads/ara_reads_2.fq \
    reads/ara_reads_3.fq > map.sam

Users can output the mapping results in PAF format by using the -outfmt paf option:

$ falign -num_threads 48 -outfmt paf ^GATC ref/ara_2_4.fa \
	reads/ara_reads_1.fq \
    reads/ara_reads_2.fq \
    reads/ara_reads_3.fq > map.paf

Some comments on usage

1. The restriction enzyme sequence

In the running commands above, ^GATC is the sequence of the DpnII restriction enzyme used for generating the reads. falign provides the sequences for all familiar restriction enzymes (to see the following information, just type the falign command) so that you don't bother to lookup for them in other places:

*** Examples of familiar <enzyme_seq>:
Enzyme_Name             Enzyme_Seq
DpnII                   ^GATC
HindIII                 A^AGCTT
NcoI                    C^CATGG
NlaIII                  CATG^

The input format of reads

Besides the way in the examples above, falign also accepts reads input in the following way:

• Read list. You can list the paths of all the reads in a file:

$ cat read_list.lst
/Users/chenying/Desktop/mwj/temp/Falign/sample-data/reads/ara_reads_1.fq
/Users/chenying/Desktop/mwj/temp/Falign/sample-data/reads/ara_reads_2.fq
/Users/chenying/Desktop/mwj/temp/Falign/sample-data/reads/ara_reads_3.fq

And then input read_list.lst to falign:

falign -num_threads 48 ^GATC ref/ara_2_4.fa.gz read_list.lst > map.sam

• Directory. If you have many FASTQs of reads in a directory, just type the name of the directory:

falign -num_threads 48 ^GATC ref/ara_2_4.fa.gz reads > map.sam

Note that only read files are allowed in the reads directory. If you put other files in the reads, falign will complain.

Output format

falign supports SAM output format:

$ falign -outfmt sam

and PAF format:

$ falign -outfmt paf

In each output result (note that every alignment has for offsets: read start, read end, reference start, reference end), falign adds the following additional fields:

• qS:i: the nearest restiction enzyme site to the read start position
• qE:i: the nearest restiction enzyme site to the read end position
• vS:i: the nearest restriction enzyme site to the reference start position
• vE:i: the nearest restriction enzyme site to the reference end position
• pi:f: percentage of identity of the alignment
• gs:i: the global chain score of the alignment's candidate
• hm:Z: a homologous map of the fragment

Use u4falign for manipulating alignment results

u4falign is used for manipulating output results of falign. It supports the following commands:

• sam2salsa2 Transfer SAM results to pairwise contacts for SALSA2
• sam23ddna Transfer SAM results to pairwise contacts for 3DDNA
• paf2salsa2 Transfer PAF results to pairwise contacts for SALSA2
• paf23ddna Transfer PAF results ot pairwise contacts for 3DDNA
• sam2frag-sam Transfer SAM results output by falign to fragment SAM mapping results

Difference between read-SAM and frag-SAM

By convention, in SAM mapping results, a read may contain multiple mapping results. For saving space, the read sequence is usually only presented in the first mapping result of this read. In the second to last mapping results of this read, the sequence field is filled by a start *. falign outputs SAM mapping results in this manner. And we call SAM mapping results output in this manner read-SAM. Since a Pore-C read always contains multiple fragments, a Pore-C read usually contains many mapping results:

0cd79600-51cf-4255-a6f7-0e9660721e85	16	2	1794202	1 ...
0cd79600-51cf-4255-a6f7-0e9660721e85	16	2	1791136	60 ...
0cd79600-51cf-4255-a6f7-0e9660721e85	0	2	1733063	60 ...
0cd79600-51cf-4255-a6f7-0e9660721e85	16	4	12727850	60 ...
0cd79600-51cf-4255-a6f7-0e9660721e85	0	2	1713207	60 ...

In the example above, the read 0cd79600-51cf-4255-a6f7-0e9660721e85 contains five alignment results.

Some tools such as whatshap taking BAM format as input will complain if there exist too many duplicate read names. In this case we suggest transfer read-SAM to frag-SAM. In frag-SAM every fragment is treadted as an individual read. We can use the sam2frag-sam command in the u4falign to transfer read-SAM to frag-SAM:

$ u4falign sam2frag-sam map.sam frag-map.sam

After transformation, we have

0cd79600-51cf-4255-a6f7-0e9660721e85_0000000001:000:0000000000:0001794201	16	2	1794202	1 ...
0cd79600-51cf-4255-a6f7-0e9660721e85_0000000001:001:0000000000:0001791135	16	2	1791136	60 ...
0cd79600-51cf-4255-a6f7-0e9660721e85_0000000001:002:0000000000:0001733062	0	2	1733063	60 ...
0cd79600-51cf-4255-a6f7-0e9660721e85_0000000001:003:0000000001:0012727849	16	4	12727850	60 ...
0cd79600-51cf-4255-a6f7-0e9660721e85_0000000001:004:0000000000:0001713206	0	2	1713207	60 ...

In frag-SAM, the sequence field in every alignment results is represented by the fragment sequence (not the whose read sequence). Note that we add a suffix to every read name to avoid duplicate read names in the SAM file. The meanings of the fields in the suffix string is

read-id:fragment-id:reference-sequence-id:reference-mapping-position

Maintainers

• Chuan-Le Xiao, [email protected]
• Ying Chen, [email protected]

Citation

License

GPLv3