在利用mothur align.seqs模块将tags比对到sivla参考序列时,为了减少计算量,通常会对silva进行区域的筛选。
针对v4区域的参考位置是start=11894, end=25319
v3-5区域的参考位置是start=6426,end=27654
v3-4区域的参考位置是start=6428, end=23444
材料参考源:
http://www.mothur.org/forum/viewtopic.php?p=7548
http://www.mothur.org/forum/viewtopic.php?f=3&t=3327&p=9623&hilit=pcr.seqs+v3_4#p9623
http://www.mothur.org/forum/viewtopic.php?f=3&t=2498&p=10536&hilit=pcr.seqs+v3_4#p10536
具体的位置界定方法:
1) Find an Ecoli 16S sequence,
2)Trim the sequence to the region within your primers. Whether or not to keep the primer sequences doesn't matter
3)Align the trimmed sequence to the reference alignment (SILVA is preferred)
4)Run summary.seqs on the aligned sequence
5)Use the start and end numbers inpcr.seqs
具体的操作实例可参考:
http://www.mothur.org/forum/viewtopic.phpf=3&t=2498&p=10536&hilit=pcr.seqs+v3_4#p10536
在实际使用中遇到以下问题:
Some of you sequences generated alignments that eliminated too many bases
针对以上情况,为了保证结果准确性,同时不严重增加计算量的情况下:
v3-4区域目前的建议是:start=1, end=25000
相关的具体原理需进一步研究
https://blog.sciencenet.cn/blog-306699-882983.html
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