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我正在尝试使用 BWA MEM 来对齐一些 WGS 文件，但我注意到一些奇怪的东西。当我过去samtools flagstat检查这些 .bam 文件时，我注意到大多数读取都未映射。

76124692 + 0 in total (QC-passed reads + QC-failed reads)
308 + 0 secondary
0 + 0 supplementary
0 + 0 duplicates
708109 + 0 mapped (0.93% : N/A)
76124384 + 0 paired in sequencing
38062192 + 0 read1
38062192 + 0 read2
0 + 0 properly paired (0.00% : N/A)
12806 + 0 with itself and mate mapped
694995 + 0 singletons (0.91% : N/A)
11012 + 0 with mate mapped to a different chr
1682 + 0 with mate mapped to a different chr (mapQ>=5)

以前，我曾经Samtofastq将我的 .bam 文件转换为 .fastq。当我头这个文件时，显示如下：

@SRR1513845.100000000/1
AACGAAACGAAAAGAAAAGAAAAGAAAGAAAAAGAAAGGAACAGAAAAG
+
AAA?=>'2&)&)&&))2(-'(,.%)&31%%'6/6,(1,501046124&6
@SRR1513845.100000000/2
AATTAATTAAGCCCCGAAGGAAGCGAGAAACACTG
+
AAA?B=AB@A@A=?A>AA@?.@?8<.1;><*17?<
@SRR1513845.100000001/1
TATAACCATATAACAAATCCAAGCCCAACAGAGAAGAGAAACAAAAAGA
+
>27<@>&856;.'.&9.%>%::-5194&:+'5);;%1&'/%%999%5(8
@SRR1513845.100000001/2
TCCAACTGATATCGTAATT
+
@3<@A>:8;?:383>=3:=
@SRR1513845.100000003/1
TATCGGTCTTGTTTAG
+
=1;=6?(4>4A13?0A
@SRR1513845.100000003/2
TTCAGGTGCCTCGAAGTTGGATAAGG
+
==>>9@;?3<A5>7);)<9-<25<9?
@SRR1513845.100000004/1
GTCATTTAGCCCAAGAGAATGGC
+
BB@ABA@@A?</A>>25A;@4:5
@SRR1513845.100000004/2
GGAGATCGAGTCAAATTTTATGCTAGGTAT
+
%A:<@7A@@=4AA?7<A5>@;3&?>>:;:>
@SRR1513845.100000012/1
GCGTCGTTATCCAAAA
+
>A:9:?88=<=0&>>9
@SRR1513845.100000012/2
TGGAAATATTTATTACCCCCCCCCCCCCCCCCCCCCCCCCC
+
A;>@A;4;=??8=:@;-4<?632;=:67;>=):9>9%88=9
@SRR1513845.100000016/1
CGTGGAATGGGGTGTGATTTAATTATCGAATGGCGTCCGATCCAGATT

这些字符 (<.@;:) 是正常的并且会影响 bwa 的对齐方式吗？

这是我的 bwa 代码：

bwa mem -M -t 38 -p hsa_GRCh38.fa SRR1513_fastqtosam.fq -o SRRR1513_aligned.bam

和我的 samtofastq 代码

java -Xmx8G -jar picard.jar SamToFastq \
I= SRR1513_fastqtosam.bam \
FASTQ= SRR1513_fastqtosam.fq \
CLIPPING_ATTRIBUTE=XT \
CLIPPING_ACTION=2 \
INTERLEAVE=true \
NON_PF=true TMP_DIR=./temp

几个小时后，我就陷入了困境。提前致谢！

更新：

我只是在 bwa mem 对齐期间注意到一个标志

[M::mem_pestat] skip orientation FF as there are not enough pairs
[M::mem_pestat] skip orientation RF as there are not enough pairs
[M::mem_pestat] skip orientation FR as there are not enough pairs
[M::mem_pestat] skip orientation RR as there are not enough pairs
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