新⼀代测序技术组装拼接软件velvet使⽤简介
⽬前⽤于新⼀代的测序的主要仪器有Illumina/Solexa的Genome Analyzer、ABI的Solid和Roche的454，它们都能⾼通量的测序，产⽣⼤量的测序结果，接下来就要对序列进⾏拼接，⽤于拼接的软件也有很多，⽐如velvet、soap、abyss、maq
等，454的还有专门的newbler。平时⽤velvet⽐较多，就简单介绍⼀下。
velvet对短序列的拼接效果⽐较好，所以多⽤于对Illumina等产⽣的短序列⽚段进⾏组装拼接。下⾯以Illumina的GAII产⽣的结果为例进⾏说明。
⼀、单端测序
单端测序可以直接对fastq格式的原始⽂件进⾏处理，⾸先是⽤velveth命令建⽴hash表⼦集
输⼊./velveth会出来使⽤帮助：
Usage:
./velveth directory hash_length {[-file_format][-read_type] filename} [options]
directory : directory name for output files
hash_length : odd integer (if even, it will be decremented) <= 75 (if above, will be reduced)
filename : path to quence file or – for standard input
File format options:
-fasta
-fastq
酷毕-
-
-eland
-gerald
Read type options:
-
short
-shortPaired
-short2
-shortPaired2
-long
-longPaired
Options:
-strand_specific : for strand specific transcriptome quencing data (default: off)
Output:
directory/Roadmaps
directory/Sequences
这⼀步主要是要确定使⽤的hash值，hash值必须为奇数，且⼩于MAXKMERLENGTH，这个值默认为31，但是在安装的时候可以调整。具体的命令可以是：
velveth result 29 -fastq -short quence.fastq
建⽴hash⼦集后⽤velvetg命令进⾏组装，输⼊./velvetg也会出来帮助：罗怎么组词
Usage:
计算机使用./velvetg directory [options]
directory : working directory name
Standard options:
-cov_cutoff <floating-point|auto> : removal of low coverage nodes AFTER tour bus or allow the system to infer it
(default: no removal)
-ins_length <integer> : expected distance between two paired end reads (default: no read pairi
ng)
-read_trkg <yes|no> : tracking of short read positions in asmbly (default: no tracking)
谷歌退出中国市场-min_contig_lgth <integer> : minimum contig length exported to contigs.fa file (default: hash length * 2)
-amos_file <yes|no> : export asmbly to AMOS file (default: no export)
-exp_cov <floating point|auto> : expected coverage of unique regions or allow the system to infer it
(default: no long or paired-end read resolution)
Advanced options:
-ins_length2 <integer> : expected distance between two paired-end reads in the cond short-read datat
(default: no read pairing)
-ins_length_long <integer> : expected distance between two long paired-end reads (default: no re
ad pairing)
食堂外包
-ins_length*_sd <integer> : est. standard deviation of respective datat (default: 10% of corresponding
length)
[replace '*' by nothing, '2' or '_long' as necessary]
-scaffolding <yes|no> : scaffolding of contigs ud paired end information (default: on)
-max_branch_length <integer> : maximum length in ba pair of bubble (default: 100)
-max_divergence <floating-point>: maximum divergence rate between two branches in a bubble (default: 0.2)
-max_gap_count <integer> : maximum number of gaps allowed in the alignment of the two branches of a
bubble (default: 3)
-
min_pair_count <integer> : minimum number of paired end connections to justify the scaffolding of two long
contigs (default: 10)
-max_coverage <floating point> : removal of high coverage nodes AFTER tour bus (default: no removal)
-long_mult_cutoff <int> : minimum number of long reads required to merge contigs (default: 2)
教学设计步骤-unud_reads <yes|no> : export unud reads in UnudReads.fa file (default: no)
Output:
directory/contigs.fa : fasta file of contigs longer than twice hash length
: stats file (tab-spaced) uful for determining appropriate coverage cutoff
directory/LastGraph : special formatted file with all the information on the final graph
directory/velvet_asm.afg : (if requested) AMOS compatible asmbly file
这⾥主要⽤到的参数是-cov_cutoff（覆盖度）这个参数，其他都可不加，其实覆盖度参数也可以省略，但是最好还是加上，增加可靠性。还有 -exp_cov这个参数加上后会对重复区域进⾏处理，也最好加上，后⾯的数值⽤auto就可以了。其他的⼀些参数就根据你⾃⼰的喜好来设置。具体的命令可以是：
velvetg result -cov_cutoff 30 -exp_cov auto -min_contig_lgth 100
最后根据出来的n50和max contig长度来判断拼接的效果，为了达到最好的拼接效果，⼀般要对hash值和覆盖度进⾏⼀系列的设置来进⾏⽐较。
⼆、双端测序卷腹
双端测序与单端测序相⽐在运⾏velveth时将-short参数改为-shortPaired，其他⼀样。
在运⾏velvetg时可加⼊-ins_length 和-ins_length_sd 参数提⾼拼接的准确性。具体命令可以是：
晋韵楼
velvetg result -cov_cutoff 30 -ins_length 300 -ins_length_sd 100 -exp_cov auto -min_contig_lgth 100
其实运⾏这⼏个命令很简单，最重要的还是根据你的测序数据选择合适的参数。
