Pins (拼接)

Scaffolding tool based on linked reads, Hi-C reads and linkage disequilibrium information.

Overview

pins is a scaffolding toolkit, it contains three basic programs, namely, pin_ld a scaffolder based on linkage disequilibrium information, pin_hic a scaffolder using Hi-C data, and pin_10x a scaffolder based on linked reads. It applies a dual selection and local optimal strategy to bridge two contigs and output a SAT file for each iteration, the SAT format is the extension of GFA format which is able to record the scaffolding process, and can also be useful for further genomic analysis. please change the text

Dependencies

zlib

Installation

Run the following commands to install pins:

git clone https://github.com/dfguan/pins.git
cd pins/src && make

Usage

Scaffolding with linked reads

Linked-read preprocessing

Given a list 10xlist of 10x read files (suppose in fastq.gz format, paired files in a line) and the assembly asm, use the following code to get read files alignments.

bwa index $asm
while read -r r1 r2
do
	prefix=`basename $r1 .fastq.gz`
	10x_trim -c -p $prefix $r1 $r2 # generate trimmed read files $prefix_{1,2}.fq.gz
	bwa mem -t12 $asm $prefix_1.fq.gz $prefix_2.fq.gz | samtools view -b - > $prefix.bam
done < $10xlist

Linked-read scaffolding

With linked-read alignment files bams and the draft assembly asm, you can run the following code build scaffolds.

samtools faidx $asm
./bin/pin_10x link $bam1 $bam2 $bam3 ... > link.matrix  # this will calcuate the numbers of shared barcode between pairs of contigs.
./bin/pin_10x build -c $asm.fai link.matrix > scaffolds.sat # this will generate scaffolding paths. 
./bin/pin_10x gets -c $asm scaffolds.sat > scaffolds.fa # this will generate scaffolds by a given SAT file.

Scaffolding with Hi-C reads

Hi-C Read preprocessing

Given a list hiclist of Hi-C read files (suppose in fastq.gz format, paired files in a line) and the assembly asm, use the following code to generate Hi-C alignment files.

bwa index $asm
while read -r r1 r2
do
	prefix=`basename $r1 .fastq.gz`
	bwa mem -SP -B10 -t12 $asm $prefix_1.fq.gz $prefix_2.fq.gz | samtools view -b - > $prefix.bam
done < $hiclist

Hi-C scaffolding

Given Hi-C reads alignment bams, a draft assembly asm and a output directory outdir, if you want to build scaffols with Hi-C in N (default: 3) rounds, please try the following commands. The final assembly will be named as scaffols_final.fa.

samtools faidx $asm 
./bin/pin_hic_it -i $N -c $asm.fai -x $ref -O $outdir $bam1 $bam2 $bam3 ...

Or you want to build scaffolds step by step:

Step 1. contact matrix calculation

From a draft assembly asm：

samtools faidx $asm
./bin/pin_hic link $bam1 $bam2 $bam3 ... > link.matrix  # this will calcuate contact numbers between any pairs of contigs.

From a sat file：

./bin/pin_hic link -s $sat $bam1 $bam2 $bam3 ... > link.matrix  # this will calcuate contact numbers between any pairs of contigs.

Step 2. Scaffolding graph construction

From a draft assembly asm:

/bin/pin_hic build -w100 -k3 -c $asm.fai link.matrix > scaffolds.sat # this will generate scaffolding paths.

From a sat file:

/bin/pin_hic build -w100 -k3 -s $sat link.matrix > scaffolds.sat # this will generate scaffolding paths.

Step 3. Mis-join detection

Given a sat file:

./bin/pin_hic break $sat $bam1 $bam2 $bam3 ... > scaffs.bk.sat
./bin/pin_hic gets -c $asm scaffs.bk.sat > scaffols_final.fa # get scaffold sequences.

A scaffolding pipeline of 3 iterations:

samtools faidx $asm
for i in `seq 1 3`
do
	if [ $i -eq 1 ]
	then 
		./bin/pin_hic link $bam1 $bam2 $bam3 ... > links_$i.matrix
		./bin/pin_hic build -w100 -k3 -c $asm.fai links_$i.matrix > scaffolds_$i.sat
	else
		./bin/pin_hic link -s scaffolds_$pi.sat $bam1 $bam2 $bam3 ... > links_$i.matrix
		./bin/pin_hic build -w100 -k3 -s scaffolds_$pi.sat links_$i.matrix > scaffolds_$i.sat 
	fi
	pi=i
done
./bin/pin_hic break -s scaffolds_$i.sat $bam1 $bam2 $bam3 ... > scaffolds_bk.sat 
./bin/pin_hic gets -c $asm scaffs.bk.sat > scaffols_final.fa

Scaffolding with linkage disequilibrium information

shall be updated soon...

Output format: SAT (V 0.1)

SAT format is extended from the GFA 1.0 format.

Record types

Tag	Description	Comment
H	Header	optional
S	Sequence	required
L	Link	optional
P	Path	optional
A	Scaffold set	optional
C	Current scaffold set	optional

`H` Header

Col	Field	Regexp	Description	Comment
1	TAG	`H`	Tag	Required
2	VER	`VN:Z:[0-9]\.[0-9]`	Version	Required

`S` Sequence

Col	Field	Regexp	Description	Comment
1	TAG	`S`	Tag	Required
2	SNAME	`.+`	Sequence name	Required, primary key
3	SLEN	`[0-9]+`	Sequence length	Required
4	SEQ	`\*\|[A-Za-z]+`	Sequence	Required

`L` Link

Col	Field	Regexp	Description	Comment
1	TAG	`P`	Tag	Required
2	SRCS	`.+`	Source sequence name	Required, foregin key S:SNAME
3	SRCE	`[-+]`	Source end	Required, `+` for 5' end and `-` for 3'
4	TGTS	`.+`	Target sequence name	Required, foregin key S:SNAME
5	TGTE	`[-+]`	Target end	Required, `+` for 5' end and `-` for 3'
6	WGT	`wt:f:[0-9]*\.?[0-9]+`	Link weight	Optional

`P` Path

Col	Field	Regexp	Description	Comment
1	TAG	`P`	Tag	Required
2	PNAME	`[cu][0-9]{9}`	Path name	Required, primary key
3	PLEN	`[0-9]+`	Path length	Required
4	NAMEL	`((.+[-+],)(.+[-+]))\|((u[0-9]{9}[-+],)u[0-9]{9}[-+])`	List of sequence names or path names	Required, foregin keys S:SNAME

`A` Scaffold set (or assembly set ?)

Col	Field	Regexp	Description	Comment
1	TAG	`A`	Tag	Required
2	ANAME	`a[0-9]{5}`	Scaffold set name	Required
3	PNAMEL	`([cu][0-9]{9},)*[cu][0-9]{9}`	List of path names	Required, foregin keys P:PNAME

`C` Current scaffold set

Col	Field	Regexp	Description	Comment
1	TAG	`C`	Tag	Required
2	CNAME	`a[0-9]{5}`	Current scaffold set name	Required, foregin key A:ANAME

Example

H	VN:Z:0.1
S	LR132056.1.4	138023	*
S	LR132056.1.5	1128790	*
S	LR132056.1.6	4496575	*
P	u000000004	662215	LR132053.1.4+,LR132053.1.5+,LR132053.1.6+
L	LR132051.1.5	+	LR132051.1.4	-	wt:f:0.028248
L	LR132051.1.6	+	LR132051.1.5	-	wt:f:0.009367
A	a00000	1	u000000004
C	a00000

Limitation

FAQ

Contact

Every one is Wellcomed to use and distribute the package. Bug report or any other suggestions, please use the github webpage or email me dfguan9@gmail.com.

dfguan/pins