featureCounts真的很厲害。
常見的參數（沒什么好說的，畢竟是固定的）：
-a -o input_file1 -F -t -g -Q -T
關鍵是以下幾個參數怎么設置：
-f # Perform read counting at feature level -O # Assign reads to all their overlapping meta-features -M # Multi-mapping reads will also be counted. --primary # Count primary alignments only. --ignoreDup # Ignore duplicate reads in read counting. -s # Perform strand-specific read counting. -p # If specified, fragments (or templates) will be counted instead of reads.
-B # Only count read pairs that have both ends aligned
-C # Do not count read pairs that have their two ends mapping
1. 什么時候需要在feature級別計數？
2. 是否要計多重比對？
3. 是否該只用最優比對？
When --primary is specified, the -M option will be ignored, meaning that a primary alignment will always be counted no matter the read is multi-mapped or not.
4. 是否要忽略重復reads？
5. pair-end模式下是否要用fragment計數？
For paired-end reads, you should count read pairs (fragments) rather than reads because counting fragments will give you more accurate counts. There are several reasons why you cannot get the fragment counts by simply dividing the counts you got from counting reads by two.
https://support.bioconductor.org/p/63824/
問題：
|| Total fragments : 706143 || || Successfully assigned fragments : 71337 (10.1%) || || Running time : 0.08 minutes ||
分配上的reads少得可憐，100%有問題！！！
因為我的unique比對率達到95%以上，怎么會這么少！！！
WARNING: reads from the same pair were found not adjacent to each || || other in the input (due to read sorting by location or || || reporting of multi-mapping read pairs).
最終查明：是注釋文件的問題！！！正常是70%。
如果沒有特別需求，STAR比對的時候就不要sort by coordinate了！！！sort一般都是為了建index
可以參考的鏈接：
不同參數的結果比較：
featureCounts -p -Q 10 -s 0 -T $cpu -a $gtf-o $out_dir/${sName}.all.counts.txt $out_dir/${sName}.hg19Aligned.out.bam
|| Threads : 1 || || Level : meta-feature level || || Paired-end : yes || || Strand specific : no || || Multimapping reads : not counted || || Multi-overlapping reads : not counted || || Min overlapping bases : 1 || || || || Chimeric reads : counted || || Both ends mapped : not required || || Features : 1199851 || || Meta-features : 58302 || || Chromosomes/contigs : 47 || || || || Paired-end reads are included. || || Assign fragments (read pairs) to features... || || || || WARNING: reads from the same pair were found not adjacent to each || || other in the input (due to read sorting by location or || || reporting of multi-mapping read pairs). || || || || Read re-ordering is performed. || || || || Total fragments : 419853 || || Successfully assigned fragments : 306852 (73.1%) || || Running time : 0.06 minutes ||
ssigned 306852 Unassigned_Unmapped 0 Unassigned_MappingQuality 0 Unassigned_Chimera 0 Unassigned_FragmentLength 0 Unassigned_Duplicate 0 Unassigned_MultiMapping 36280 Unassigned_Secondary 0 Unassigned_Nonjunction 0 Unassigned_NoFeatures 56950 Unassigned_Overlapping_Length 0 Unassigned_Ambiguity 19771
featureCounts -p -Q 10 -M -s 0 -T $cpu -a $gtf -o $out_dir/${sName}.all.counts.txt2 $out_dir/${sName}.hg19Aligned.out.bam
|| Threads : 1 || || Level : meta-feature level || || Paired-end : yes || || Strand specific : no || || Multimapping reads : counted || || Multi-overlapping reads : not counted || || Min overlapping bases : 1 || || || || Chimeric reads : counted || || Both ends mapped : not required || || || \\===================== http://subread.sourceforge.net/ ======================// //================================= Running ==================================\\ || || || Features : 1199851 || || Meta-features : 58302 || || Chromosomes/contigs : 47 || || || || Paired-end reads are included. || || Assign fragments (read pairs) to features... || || || || WARNING: reads from the same pair were found not adjacent to each || || other in the input (due to read sorting by location or || || reporting of multi-mapping read pairs). || || || || Read re-ordering is performed. || || || || Total fragments : 419853 || || Successfully assigned fragments : 306852 (73.1%) || || Running time : 0.05 minutes ||
Assigned 306852 Unassigned_Unmapped 0 Unassigned_MappingQuality 36280 Unassigned_Chimera 0 Unassigned_FragmentLength 0 Unassigned_Duplicate 0 Unassigned_MultiMapping 0 Unassigned_Secondary 0 Unassigned_Nonjunction 0 Unassigned_NoFeatures 56950 Unassigned_Overlapping_Length 0 Unassigned_Ambiguity 19771
featureCounts -p -Q 10 -B -s 0 -T $cpu -a $gtf -o $out_dir/${sName}.all.counts.txt3 $out_dir/${sName}.hg19Aligned.out.bam
|| Threads : 1 || || Level : meta-feature level || || Paired-end : yes || || Strand specific : no || || Multimapping reads : not counted || || Multi-overlapping reads : not counted || || Min overlapping bases : 1 || || || || Chimeric reads : counted || || Both ends mapped : required || || || \\===================== http://subread.sourceforge.net/ ======================// //================================= Running ==================================\\ || || || Features : 1199851 || || Meta-features : 58302 || || Chromosomes/contigs : 47 || || Paired-end reads are included. || || Assign fragments (read pairs) to features... || || || || WARNING: reads from the same pair were found not adjacent to each || || other in the input (due to read sorting by location or || || reporting of multi-mapping read pairs). || || || || Read re-ordering is performed. || || || || Total fragments : 419853 || || Successfully assigned fragments : 306500 (73.0%) || || Running time : 0.05 minutes ||
Assigned 306500 Unassigned_Unmapped 656 Unassigned_MappingQuality 0 Unassigned_Chimera 0 Unassigned_FragmentLength 0 Unassigned_Duplicate 0 Unassigned_MultiMapping 36133 Unassigned_Secondary 0 Unassigned_Nonjunction 0 Unassigned_NoFeatures 56838 Unassigned_Overlapping_Length 0 Unassigned_Ambiguity 19726
featureCounts -p -Q 10 --ignoreDup -s 0 -T $cpu -a $gtf -o $out_dir/${sName}.all.counts.txt4 $out_dir/${sName}.hg19Aligned.out.bam
|| Threads : 1 || || Level : meta-feature level || || Paired-end : yes || || Strand specific : no || || Multimapping reads : not counted || || Multi-overlapping reads : not counted || || Min overlapping bases : 1 || || Duplicated Reads : ignored || || || || Chimeric reads : counted || || Both ends mapped : not required || || || \\===================== http://subread.sourceforge.net/ ======================// //================================= Running ==================================\\ || || || Features : 1199851 || || Meta-features : 58302 || || Chromosomes/contigs : 47 || || || || Paired-end reads are included. || || Assign fragments (read pairs) to features... || || || || WARNING: reads from the same pair were found not adjacent to each || || other in the input (due to read sorting by location or || || reporting of multi-mapping read pairs). || || || || Read re-ordering is performed. || || || || Total fragments : 419853 || || Successfully assigned fragments : 306852 (73.1%) || || Running time : 0.05 minutes ||
Assigned 306852 Unassigned_Unmapped 0 Unassigned_MappingQuality 0 Unassigned_Chimera 0 Unassigned_FragmentLength 0 Unassigned_Duplicate 0 Unassigned_MultiMapping 36280 Unassigned_Secondary 0 Unassigned_Nonjunction 0 Unassigned_NoFeatures 56950 Unassigned_Overlapping_Length 0 Unassigned_Ambiguity 19771