Closed chongjing closed 3 months ago
The reference look to be the same.
Can you confirm that the BAM file has reads?
What does the output in $F.ref.chrA.list
look like?
Unfortunately the octoploid analysis was never extensively tested. I will do my best to help you through your analysis though. We might have to trace backwards to see where reads are lost.
Thanks so much for quick reply.
The reference look to be the same.
I would say they are not the same, as the reads are aligned to chr[A|B|C|D].exon.fa
which are basically genes. But the refseq.chrA.gtf
is annotation for whole genome.
Can you confirm that the BAM file has reads? What does the output in
$F.ref.chrA.list
look like?Unfortunately the octoploid analysis was never extensively tested. I will do my best to help you through your analysis though. We might have to trace backwards to see where reads are lost.
It seems the BAM file is fine:
V300110184L2C006R0410597611 129 g2.t1 162 0 128M1D22M g42147.t1 140 0 AGGGGAGACTACTCCTCCGTCGTCGCGGTTGTCGGAGGGCTCGTCCGGTCTTAGCAGGTCGCTGTCGCGGTCCATTCTCTCGCCGAAGGTGTCGACTGCGTATATATCGTCTAGGGCTGCTACGATTGCATGGCTTTCTCCTCTTCTTCC FFEFFDFEFFFFFFFFFFFFFFAFFFFF?EFFCFF8FEFEFFFFFFFCEFFEFFDFFFDFFFFFDFEEFFFFFFDFFFFFFFFFFFFFFFFEFF9FFFDFFFBFFFFFFFEDFFFFFFFFFF@F<FFFEFFFFFFFEFFDFDEFCEFFFF AS:i:-63 XN:i:0 XM:i:11 XO:i:1 XG:i:1 NM:i:12 MD:Z:128^C2G2A1G0G1A0T0G3G0G0G1G1 YT:Z:UP
V300110184L2C003R0130387205 153 g3.t1 6 42 150M = 6 0 TTTTGTTGGGGATATCGATGCGGTGGTGCCGGAGCTAATTCTTACAGTCCATGACGGGAAGGGTGTATATGTGCAAATTGATGATTACTCTGGATCACAAGAGCCGTCTTCAACTTGTTCTTGTTGGAAAAGATGGAACGTCTGGCGGTG FGFGFGFGGFFFFFFGFGFFFGFGFGFEGFGGGFFFFGFFFGGFGGGFFFFFGFGGGFFFGGFDGGFGFFFGFFFFGFFG@FFFGEFGFGGGFFFEFFFFFGFFFGFFFFFGFGFGFFFFFFF>FGGFFFFFFFGFFFFFFFGGFGF>GG AS:i:0 XN:i:0 XM:i:0 XO:i:0 XG:i:0 NM:i:0 MD:Z:150 YT:Z:UP
V300110184L2C002R0530103382 153 g3.t1 8 42 150M = 8 0 TTGTTGGGGATATCGATGCGGTGGTGCCGGAGCTAATTCTTACAGTCCATGACGGGAAGGGTGTATATGTGCAAATTGATGATTACTCTGGATCACAAGAGCCGTCTTCAACTTGTTCTTGTTGGAAAAGATGGAACGTCTGGCGGTGGG EGGFFFFFGFGGEGGFGFFFGGFFFGFGGFGDGGFFGFFGGGGEFFGGFGGFGGFFGGFGFGFBFGFFGGFGFFGFGFGGFFGFFFFFFGFFFGFFFFFFFFGGFFGEFFFGFGGFFGFFFFGGFFFGGGG@GGFFGFFFEGFGFFFFFG AS:i:0 XN:i:0 XM:i:0 XO:i:0 XG:i:0 NM:i:0 MD:Z:150 YT:Z:UP
And $F.ref.chrA.list
also looks fine:
V300110184L2C002R0040539179 UNK g50383.t1 105 -8.998771 -18.616749 -92.152931 -
V300110184L2C006R0210223877 REF g31588.t1 462 -0.057198 -8.647595777539312 -92.141655 -
V300110184L2C001R0020390451 UNK g74259.t1 180 -0.100707 -9.718684 -92.170944 -
V300110184L2C001R0491032017 REF g62117.t1 155 -18.648333 -22.88515760903499 -92.988495 -
V300110184L2C003R0230789834 UNK g50383.t1 266 1.176992 -18.058962 -184.344871 -
V300110184L2C003R0141263402 REF g20128.t1 996 -14.712079 -30.368663422663783 -184.433258 -
V300110184L2C002R0361309029 UNK g31391.t1 200 -0.063814 -9.681791 -92.146133 -
V300110184L2C005R0560714001 REF g31767.t1 807 -33.012351 -50.68906171133189 -185.26374 -
Some update: I made a 'fake' annotation gtf, with genes as 'chromosomes', like this:
g1.t1 . transcript 1 615 . + . transcript_id "g1.t1";
g1.t2 . transcript 1 579 . + . transcript_id "g1.t2";
g2.t1 . transcript 1 1566 . + . transcript_id "g2.t1";
g3.t1 . transcript 1 879 . + . transcript_id "g3.t1";
g4.t1 . transcript 1 213 . + . transcript_id "g4.t1";
g5.t1 . transcript 1 435 . + . transcript_id "g5.t1";
and this works, I got reads assigned to genes from featureCounts
, $F.chrA.counts.txt
looks like this:
# Program:featureCounts v2.0.2; Command:"featureCounts" "-T" "8" "-t" "transcript" "-g" "transcript_id" "-p" "--countReadPairs" "-a" "0.chrHap1.gtf" "-o" "A_G_1.chrHap1.counts.txt" "A_G_1.chrHap1.1.ref.bam"
Geneid Chr Start End Strand Length A_G_1.chrHap1.1.ref.bam
g1.t1 g1.t1 1 615 + 615 0
g1.t2 g1.t2 1 579 + 579 0
g2.t1 g2.t1 1 1566 + 1566 0
g3.t1 g3.t1 1 879 + 879 28
g4.t1 g4.t1 1 213 + 213 0
g5.t1 g5.t1 1 435 + 435 0
g6.t1 g6.t1 1 156 + 156 0
g7.t1 g7.t1 1 153 + 153 0
g8.t1 g8.t1 1 924 + 924 81
g9.t1 g9.t1 1 681 + 681 0
g10.t1 g10.t1 1 768 + 768 0
g11.t2 g11.t2 1 5847 + 5847 0
g11.t1 g11.t1 1 4317 + 4317 0
g12.t1 g12.t1 1 435 + 435 0
g13.t1 g13.t1 1 1950 + 1950 0
g14.t1 g14.t1 1 1134 + 1134 0
g15.t1 g15.t1 1 1056 + 1056 0
g15.t2 g15.t2 1 1053 + 1053 108
g15.t3 g15.t3 1 1092 + 1092 130
g16.t1 g16.t1 1 900 + 900 24
g17.t1 g17.t1 1 819 + 819 0
g18.t1 g18.t1 1 1041 + 1041 39
g18.t2 g18.t2 1 1038 + 1038 74
Please correct me if I am in wrong direction.
Best, Chongjing
Ah I see. Looking at the workflow, it defaults to alignment to the transcriptome using bowtie. The other option, which is commented out, uses STAR to align with splicing to the genome. I'm not sure why I left bowtie as the default as I believe alignment to the genome is what I used in all other workflows.
I will change the workflow to default to using STAR genome alignment.
Sounds great. Thanks.
https://github.com/tony-kuo/eagle/blob/d6fb2e97be8515e2f7057a7d5f613bab65848d77/scripts/octoploid_analysis.sh#L162
Hi, @tony-kuo
After running your octoploid analysis pipeline, I got an issue at the very last step: I got 0% of reads successfully assigned to specific subgenome.
I realize this
eagle/$F.chrA.ref.bam
file was generated from previouseagle-rc --refonly --readlist -a chrA/$F.refsort.bam -o eagle/$F.chrA eagle/$F.ref.chrA.list
step, and it seems the reference ofeagle/$F.chrA.ref.bam
is the transcriptome of chrA. However, in your featureCounts codefeatureCounts -T 8 -t exon -g transcript_id -a refseq.chrA.gtf -o eagle/$F.chrA.counts.txt eagle/$F.chrA.ref.bam
, the reference of the annotation filerefseq.chrA.gtf
is genome of chrA. For example, header ofeagle/$F.chrA.ref.bam
looks like:while header of
refseq.chrA.gtf
looks like:My question is, does that mean the
eagle/$F.chrA.ref.bam
and therefseq.chrA.gtf
use different reference? is this the reason why I got nothing assigned to subgenome? How to solve this? Please let me know if you need more information. ThanksChongjing