具體報(bào)錯(cuò)信息如下：

[root@localhost pool-5.2]#  /home/packages/stacks-2.54/process_radtags -P -p ./raw/ -b ./pool-5.2.txt -o ./samples/  -c -q -r --inline_index --renz_1 mluCI --renz_2 avaII
Processing paired-end data.
Using Phred+33 encoding for quality scores.
Found 1 paired input file(s).
Searching for single-end, inlined and paired-end, indexed barcodes.
' (filenames can consist of letters, numbers, '.', '-' and '_').

這個(gè)錯(cuò)誤在網(wǎng)上找不到任何線索，只能通過閱讀源碼來解決:
首先定位到stacks的安裝目錄
我的目錄如下：

[root@localhost pool-5.2]# cd /home/packages/stacks-2.54/
[root@localhost stacks-2.54]# ls
acinclude.m4  ChangeLog     config.h     config.status  cstacks  INSTALL      libcore.a  Makefile     phasedstacks     process_shortreads  src       tests
aclocal.m4    clone_filter  config.h.in  configure      gstacks  kmer_filter  libpop.a   Makefile.am  populations      README              sstacks   tsv2bam
autogen.sh    config        config.log   configure.ac   htslib   libclean.a   LICENSE    Makefile.in  process_radtags  scripts             stamp-h1  ustacks
[root@localhost stacks-2.54]# 

進(jìn)入src文件夾，這個(gè)是stacks的源碼所在位置
然后搜索錯(cuò)誤信息：' (filenames can consist of letters, numbers, '.', '-' and '_').

[root@localhost stacks-2.54]# cd src
[root@localhost src]# grep -r "' (filenames can consist of letters, numbers, '.', '-' and '_')." .
./file_io.cc:                    cerr << "Invalid filename on line " << line_num << ": '" << s << "' (filenames can consist of letters, numbers, '.', '-' and '_').\n";
Binary file ./file_io.o matches
[root@localhost src]# 

注意符號不能寫錯(cuò)，用雙引號""將出錯(cuò)信息擴(kuò)起來，最后一個(gè)點(diǎn)代表在當(dāng)前目錄搜索所有文件，看看哪個(gè)文件包含錯(cuò)誤信息
可以看到與該錯(cuò)誤信息相關(guān)的文件名是file_io.cc
那我們開始查看這段代碼：

//
        // Check for the existence of a file name to associate with this barcode set.
        //
        if (q - p < id_len)
            q++;
        s = q;
        while (*q != '\0') {
            if (!isalnum(*q)) {
                switch (*q) {
                case '-':
                case '_':
                case '.':
                    break;
                case '\r':
                case '\t':
                    *q = '\0';
                    break;
                default:
                    cerr << "Invalid filename on line " << line_num << ": '" << s << "' (filenames can consist of letters, numbers, '.', '-' and '_').\n";
                    exit(1);
                }
            }
            if (*q != '\0') q++;
        }

        barcodes.push_back(BarcodePair(p, r, s));
        if (p != NULL && strlen(p) > 0) se_bc.insert(string(p));
        if (r != NULL && strlen(r) > 0) pe_bc.insert(string(r));
    }

    fh.close();

這段代碼的注釋是：// Check for the existence of a file name to associate with this barcode set.
說明這段代碼的功能是處理文件名和barcodes的關(guān)系，那么我們?nèi)菀拙拖氲?，是不是barcodes文件的命名有問題
檢查barcodes文件的所有字符，與正常的barcodes文件作為對比

[root@localhost pool-5.2]# cat -A pool-5.2.txt /有問題的barcode文件
GTCGA^IGGCTAC^IKE4^M$
TACCG^IGGCTAC^IKE5^M$
TACGT^IGGCTAC^IKE6^M$
TAGTA^IGGCTAC^IKE7^M$
TATAC^IGGCTAC^IKE8^M$
TCACG^IGGCTAC^IKE9^M$
TCAGT^IGGCTAC^IKE10^M$
TCCGG^IGGCTAC^IKE11^M$
TCTGC^IGGCTAC^IKE12^M$
TGGAA^IGGCTAC^IKE13^M$
TTACC^IGGCTAC^IJM1 ^M$

[root@localhost pool-5.2]# cat -A pool-5.2.txt /正常的barcode文件
TACCG^ITGACCA^IHJ2-13^M$
TACGT^ITGACCA^IHJ2-14^M$
TAGTA^ITGACCA^IHJ2-15^M$
TATAC^ITGACCA^IHJ2-16^M$
TCACG^ITGACCA^IXY1^M$
TCAGT^ITGACCA^IXY2^M$
TCCGG^ITGACCA^IXY3^M$
TCTGC^ITGACCA^IXY4^M$
TGGAA^ITGACCA^IXY5^M$
TTACC^ITGACCA^IXY6^M$

我們發(fā)現(xiàn)出問題barcods文件最后一行多了一個(gè)空格字符，如果直接打開是不容易看出來的，因此要使用 cat -A 把所有字符顯示出來
檢查到問題所在，我們對有問題的barcodes文件的空格刪掉

[root@localhost pool-5.2]# sed -i "s/ //g" pool-5.2.txt
[root@localhost pool-5.2]# cat -A pool-5.2.txt
GGATA^IGGCTAC^IKD8^M$
GGCCA^IGGCTAC^IKD9^M$
GGCTC^IGGCTAC^IKE1^M$
GTAGT^IGGCTAC^IKE2^M$
GTCCG^IGGCTAC^IKE3^M$
GTCGA^IGGCTAC^IKE4^M$
TACCG^IGGCTAC^IKE5^M$
TACGT^IGGCTAC^IKE6^M$
TAGTA^IGGCTAC^IKE7^M$
TATAC^IGGCTAC^IKE8^M$
TCACG^IGGCTAC^IKE9^M$
TCAGT^IGGCTAC^IKE10^M$
TCCGG^IGGCTAC^IKE11^M$
TCTGC^IGGCTAC^IKE12^M$
TGGAA^IGGCTAC^IKE13^M$
TTACC^IGGCTAC^IJM1^M$

可以看到空格已經(jīng)被刪掉了
最后我們再來運(yùn)行一下

[root@localhost pool-5.2]# /home/packages/stacks-2.54/process_radtags -P -p ./raw/ -b ./pool-5.2.txt -o ./samples/  -c -q -r --inline_index --renz_1 mluCI --renz_2 avaII
Processing paired-end data.
Using Phred+33 encoding for quality scores.
Found 1 paired input file(s).
Searching for single-end, inlined and paired-end, indexed barcodes.
Loaded 48 barcodes (5bp / 6bp).
Will attempt to recover barcodes with at most 1 / 1 mismatches.
Processing file 1 of 1 [52_R1_001.fastq.gz]
  Reading data from:
  ./raw/52_R1_001.fastq.gz and
  ./raw/52_R2_001.fastq.gz
  Processing RAD-Tags...

好了，沒有' (filenames can consist of letters, numbers, '.', '-' and '_').
問題解決！